WO2020244570A1 - 不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用 - Google Patents

不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用 Download PDF

Info

Publication number
WO2020244570A1
WO2020244570A1 PCT/CN2020/094308 CN2020094308W WO2020244570A1 WO 2020244570 A1 WO2020244570 A1 WO 2020244570A1 CN 2020094308 W CN2020094308 W CN 2020094308W WO 2020244570 A1 WO2020244570 A1 WO 2020244570A1
Authority
WO
WIPO (PCT)
Prior art keywords
lys
pyro
photosensitizer
glu
pharmaceutically acceptable
Prior art date
Application number
PCT/CN2020/094308
Other languages
English (en)
French (fr)
Inventor
洪章勇
黄伟强
李双
Original Assignee
康宏耀源（天津）科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 康宏耀源（天津）科技有限公司 filed Critical 康宏耀源（天津）科技有限公司
Publication of WO2020244570A1 publication Critical patent/WO2020244570A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/409—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. porphine derivatives, bilirubin, biliverdine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0036—Porphyrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D519/00—Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/07—Optical isomers

Definitions

  • the present application relates to a photosensitizer with tumor targeting, a preparation method thereof, and application in tumor photodynamic therapy and tumor imaging diagnosis.
  • folic acid (FA) as a tumor targeting ligand is connected to a porphine compound as a photosensitive moiety (PS) through a linking moiety of different chain lengths optionally having acidic amino acid fragments.
  • PS photosensitive moiety
  • Photodynamic therapy is a new type of local and low-toxic tumor treatment program.
  • the photosensitizer used in photodynamic therapy has low dark toxicity, and the direct action site of photodynamic therapy is the diseased tissue at the part of light radiation. Only when a certain excitation wavelength is used to irradiate the diseased part, the photosensitizer will be activated to produce killing ability.
  • the optical properties of photosensitizers determine that after being enriched in tumor tissues, they can produce obvious fluorescent signals. Therefore, they can integrate diagnosis and treatment and provide guidance for clinical surgical procedures.
  • the current photosensitizers are mainly porphine compounds.
  • the absorption wavelength of porphin compounds is about 680nm, such as: pyropheophorbide a (Pyro), methyl pyrophorbide alkyl ether derivatives (HPPH), chlorin e6 (Ce6), verteporfin ( BPD-MA, vertoporfin) and Temoporfin (Temoporfin, trade name: Foscan) and so on.
  • the extinction coefficient of Pyropheophorbide a (Pyropheophorbide a, Pyro) at a wavelength of 668nm is 3.79 ⁇ 10 4 L mol -1 cm -1 , and it has a strong singlet oxygen quantum yield (0.52), so it has Strong cell photodynamic killing activity.
  • Pyro has poor water solubility and basically has no tumor localization ability. Its low concentration in tumor tissues and high concentration in normal tissues will cause severe phototoxic side effects on normal tissues during photodynamic therapy. . Therefore, through structural modification of Pyro to solve the shortcomings that limit its clinical application, it is expected to develop the required photosensitizer.
  • the photosensitizer By coupling a photosensitizer to a targeting ligand that expresses receptors highly on the tumor surface, the photosensitizer can be given the ability to actively target the tumor, which has become an effective strategy for preparing tumor-specific photosensitizers.
  • Folic acid receptors are generally highly expressed on the surface of most tumor cells, and small molecule folic acid can be used as an ideal tumor targeting ligand and is widely used in tumor imaging and treatment.
  • the tumor-targeting group folic acid is connected to Pyro via cysteine to improve the tumor enrichment ability of Pyro.
  • the prepared compound shows a good selective ability and a very effective photodynamic therapy effect on subcutaneous tumors with high folic acid expression.
  • the molecular weight of polyethylene glycol used as a linking chain for PEGylation is the average molecular weight.
  • the molecular structure is not fixed, and the molecular weight is not fixed. It is difficult to purify the compound, and it is still not certain that the photosensitive drug molecule has excellent tumor specificity.
  • the specific structure of the enrichment function and efficient photosensitive activation and killing ability Therefore, there is still an urgent need for photosensitizers with excellent tumor targeting and improved metabolic distribution characteristics in vivo.
  • the application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof:
  • a and C are independently selected from the folic acid group (FA) and the porphine-type photosensitive moiety (PS), and when any one of A and C is the folic acid group, the other is the porphine-type photosensitive moiety;
  • L is independently selected from L1 or L2;
  • B represents an acidic amino acid fragment
  • n is an integer of 1-5;
  • o 0 or 1
  • p is an integer of 4-8.
  • this application provides a method for preparing a photosensitizer of general formula (I), which includes:
  • Lys, L, m, B, o, FA and PS are as defined above.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
  • the present application provides an imaging diagnostic agent comprising a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof.
  • this application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or its pharmaceutical composition in the preparation of a medicament for the treatment of tumors.
  • this application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or pharmaceutical composition in the preparation of a tumor imaging diagnostic agent.
  • the application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a medicament for treating tumors by photodynamic therapy.
  • the present application provides a method for treating tumors, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a method for imaging a tumor for diagnosis, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a method for treating tumors by photodynamic therapy, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for treating tumors.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for imaging tumors for diagnosis.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for treating tumors by photodynamic therapy.
  • the application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or pharmaceutical composition in the treatment of tumors.
  • the present application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in imaging tumors for diagnosis.
  • the application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the treatment of tumors by photodynamic therapy.
  • Figure 1 is a schematic diagram of the synthesis method of L1.
  • Figure 4 is a schematic diagram of the synthesis method of Pyro-Lys-(L2) 3 -Glu-FA.
  • Figure 5 is a schematic diagram of the synthesis method of Pyro-Lys-(L1) 3 -Asp-FA.
  • 8A to 8D are the ultraviolet absorption spectra of Pyro-Lys-(L1) 3 -Glu-FA and Pyro in different solutions and the singlet oxygen quantum yield measurement diagrams in PBS solution.
  • Figure 10 shows the selective endocytosis of Pyro, Pyro-Lys-(L1) 3 -Glu-FA and Temoporfin in KB and A549 cells, respectively.
  • Figure 11 shows the competitive inhibition of KB cell line activity by Pyro, Pyro-Lys-(L1) 3 -Glu-FA and Temoporfin, respectively, and folic acid.
  • Figure 12 shows the biological activity and dark toxicity of Pyro, Pyro-Lys-(L1) 3 -Glu-FA and Temoporfin at the level of different cell lines.
  • Figure 14 shows the drug metabolism process of Pyro-Lys-(L2) 3 -Glu-FA and Pyro-Lys-(L1) 3 -Asp-FA in KB transplanted tumor mice over time.
  • Figures 15A to 15E show the drug metabolism process of Pyro, Pyro-Lys-(L1) 3- Glu-FA and Temoporfin in KB, A431 and NCI-N87 transplanted tumor mice over time and their effects Enrichment of various organ tissues, tumor tissues and muscle tissues near tumors (Ht: heart; Lv: liver; Sp: spleen; Lu: lung; Kn: kidney; Tm: tumor; Ms; muscle).
  • Figures 16A to 16C show the tumor photodynamic therapy effects of Pyro and Pyro-Lys-(L1) 3 -Glu-FA on KB, A431 and NCI-N87 transplanted tumor mice, respectively.
  • Figure 17 shows the acute toxicity of Pyro-Lys-(L1) 3 -Glu-FA and Pyro to normal mice.
  • the application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof:
  • a and C are independently selected from the folic acid group (FA) and the porphine-type photosensitive moiety (PS), and when any one of A and C is the folic acid group, the other is the porphine-type photosensitive moiety;
  • L represents a linker, which is independently selected from L1 or L2;
  • B represents an acidic amino acid fragment
  • n is an integer of 1-5;
  • o 0 or 1
  • p is an integer of 4-8.
  • the folate group preferably has the following structure:
  • the porphin-type photosensitive moiety is independently selected from Pyro groups, Ce6 groups, HPPH groups and Vertoporfin groups, preferably Pyro groups or Ce6 groups , More preferably a group of Pyro.
  • the group of Pyro has the structure
  • the acidic amino acid fragments are glutamic acid fragments and aspartic acid fragments; preferably, the glutamic acid fragments have the structure And the aspartic acid fragment has the structure
  • m is an integer of 1-5; more preferably 2, 3, 4, or 5, still more preferably 3 or 5; most preferably 3.
  • o is preferably 1.
  • the number p of the repeating unit -CH 2 CH 2 O- in L2 can also be 4, 5, 6, 7, or 8, preferably 6, that is, L2 has the structure
  • -(L) m -(B) o - has one of the following structures:
  • the photosensitizer of general formula (I) is preferably selected from one of the following compounds or a pharmaceutically acceptable salt thereof:
  • the pharmaceutically acceptable salt of the photosensitizer of the general formula (I) includes a pharmaceutically acceptable acid addition salt or a pharmaceutically acceptable base addition salt, which is obtained by making the general formula (I) )
  • the photosensitizer is obtained by reacting with a pharmaceutically acceptable inorganic acid or organic acid or with a pharmaceutically acceptable inorganic base or organic base.
  • this application provides a method for preparing a photosensitizer of general formula (I), which includes:
  • Lys, L, m, B, o, FA and PS are as defined above.
  • the linker L is independently selected from L1 or L2 as defined above, where L1 can be obtained by the synthesis method described below, and L2 can be obtained by the synthesis method described below or commercially available .
  • step 2) the synthetic route from step 2) to step 5) is shown below, where three linkers L1 are used as examples of -(L) m -, and glutamic acid fragments are used as examples of acidic amino acid fragments , Using Pyro as an example of porphine-type photosensitive compound
  • the product ie, the photosensitizer of general formula (I)
  • the active ester of the porphin-type photosensitive compound PS such as the active ester of Pyro, such as Pyro-Lys-L1-FA, Pyro-Lys- (L1) 2 -FA, Pyro-Lys-(L1) 3 -FA, Pyro-Lys-(L1) 4 -FA, Pyro-Lys-(L1) 5 -FA, Pyro-Lys-(L1) 5 -Glu -FA, Pyro-Lys-(L1) 3 -Glu-FA, Pyro-Lys-(L2) 3 -Glu-FA and Pyro-Lys-(L1) 3 -Asp-FA.
  • the product was analyzed for purity by HPLC, and molecular weight was characterized by HRMS.
  • the present application provides a method for preparing the above-mentioned photosensitizer, and the detailed synthetic route is shown in Figs. 1 to 5.
  • the application provides a pharmaceutical composition
  • a pharmaceutical composition comprising a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable excipient includes any and all solvents, dispersion media, antibacterial agents, preservatives, isotonic agents, pyrogen-free water, physiological saline, and phosphate buffer solutions.
  • dispersion media include any and all solvents, dispersion media, antibacterial agents, preservatives, isotonic agents, pyrogen-free water, physiological saline, and phosphate buffer solutions.
  • auxiliary materials may be included, for example, auxiliary materials commonly used in the art.
  • the administration of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof disclosed in the present application can be carried out by any acceptable mode of administration for medicaments with similar functions, including but not limited to oral administration and Injection administration, such as subcutaneous, intravenous, topical, transdermal, intraperitoneal and intramuscular administration. Oral administration and parenteral administration are generally used to treat the indications of the subject as the preferred embodiment.
  • the photosensitizer of general formula (I) as described above or a pharmaceutically acceptable salt thereof can be formulated into a pharmaceutical composition for the treatment of tumors.
  • Standard pharmaceutical formulation techniques are used, such as those disclosed in Remington's The Science and Practice of Pharmacy, 21st Ed., Lippincott Williams & Wilkins (2005), which is incorporated herein by reference in its entirety.
  • the present application provides an imaging diagnostic agent comprising a photosensitizer of general formula (I) or a pharmaceutically acceptable salt thereof.
  • this application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or its pharmaceutical composition in the preparation of a medicament for the treatment of tumors.
  • this application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or pharmaceutical composition in the preparation of a tumor imaging diagnostic agent.
  • the application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a medicament for treating tumors by photodynamic therapy.
  • the present application provides a method for treating tumors, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a method for imaging a tumor for diagnosis, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a method for treating tumors by photodynamic therapy, which comprises administering a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof to an individual in need.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for treating tumors.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for imaging tumors for diagnosis.
  • the present application provides a photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof for treating tumors by photodynamic therapy.
  • the application provides the use of the photosensitizer of general formula (I) or its pharmaceutically acceptable salt or pharmaceutical composition in the treatment of tumors.
  • the present application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in imaging tumors for diagnosis.
  • the application provides the use of the photosensitizer of general formula (I) or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the treatment of tumors by photodynamic therapy.
  • the tumor is a cancer with high expression of folate receptor, including but not limited to oral epidermoid carcinoma, epidermal squamous cell carcinoma and gastric cancer.
  • a series of linking parts with different chain lengths optionally having acidic amino acid fragments such as Glu fragments and Asp fragments, are designed and synthesized, and folic acid, which is a tumor-targeting ligand, is combined with Pyro
  • the porphine-type photosensitive moiety of the compound is connected to obtain the photosensitizer of general formula (I), and then the photophysical properties of the photosensitizer and the biological activity evaluation in vitro and in vivo are carried out.
  • the inventors of the present application have discovered through research that the introduction of more hydrophilic linking moieties and more carboxyl functional groups can significantly improve the water solubility of porphine-type photosensitive compounds such as Pyro, and that increasing the water solubility of drug molecules can improve the drug The enrichment capacity of tumor sites and reduce the background absorption of drugs in mice.
  • the photosensitizer of the general formula (I) of the present application has at least one of the following advantages: it shows good tumor selectivity; it has high folate receptor The expressed tumor exhibits excellent tumor-specific enrichment ability and tumor cell killing ability; rapid enrichment and rapid elimination can be achieved; and toxic side effects to normal tissues can be reduced.
  • the photosensitizer of the general formula (I) of the present application has good application prospects in the field of tumor photodynamic therapy and tumor imaging diagnosis, and has the potential to be developed into drugs, especially having 3 linkers (preferably, 3 A photosensitizer of general formula (I) with a total molecular weight of about 1K) and a linking part of an acidic amino acid fragment, such as Pyro-Lys-(L1) 3 -Glu-FA, Pyro-Lys-(L2) 3- Glu-FA and Pyro-Lys-(L1) 3- Asp-FA have the potential to be developed into drugs.
  • 3 linkers preferably, 3 A photosensitizer of general formula (I) with a total molecular weight of about 1K
  • a linking part of an acidic amino acid fragment such as Pyro-Lys-(L1) 3 -Glu-FA, Pyro-Lys-(L2) 3- Glu-FA and Pyro-Lys-(L1) 3- Asp-FA have the
  • PDT photodynamic therapy
  • Pyro pyropheophorbide a
  • FA folic acid
  • Lys lysine
  • Glu glutamic acid
  • Asp aspartic acid
  • PEG polyethylene glycol
  • Et 2 O Diethyl ether
  • THF tetrahydrofuran
  • Et 3 N triethylamine
  • DIEA N,N-diisopropylethylamine
  • EA ethyl acetate
  • DCM dichloromethane
  • EtOH ethanol
  • MeOH methanol
  • DMF N , N-dimethylformamide
  • DMSO dimethyl sulfoxide
  • HCl hydrochloric acid
  • NaOH sodium hydroxide
  • NaHCO 3 sodium bicarbonate
  • Na 2 SO 4 sodium sulfate
  • MgSO 4 magnesium sulfate
  • Pd/ C Palladium/Carbon
  • H 2 Hydrogen
  • TFA Trifluoride
  • Pyro was purchased from Ningbo Dongmi Biological Technology Co., Ltd.; FA, diglycolic anhydride, EDC, NHS and TFA were purchased from Tianjin Ciense Biochemical Technology Co., Ltd.; Glu, Lys, Cbz-Cl, Fmoc-OSu, triphenyl chloride Resin, HATU, and HoBt were purchased from Gill Biochemical (Shanghai) Co., Ltd.; 4,7,10-trioxo-1,13-tridecanediamine and Tris were purchased from Tixiai (Shanghai) Chemical Industry Development Co., Ltd.; DIEA Purchased from Beijing Bailingwei Technology Co., Ltd.; Fmoc-PEG 6 -CH 2 CH 2 COOH was purchased from Chengdu Furuikang Biotechnology Co., Ltd.; Pd/C was purchased from Xi'an Kaili Chemical Co., Ltd.; CO 2 and H 2 were purchased from Tianjin Liufang Industrial Gas Distribution Co., Ltd.
  • triphenyl chloride resin (5.0 mmol) with a loading of 1.0 mmol/g was swelled with dichloromethane.
  • the Fmoc protecting group was removed with 20% piperidine (PIP) in DMF for 30 minutes, and eluted with DMF 6 times.
  • the synthesized L1 1.5eq
  • the condensing agent O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) 2.0eq
  • 1-hydroxybenzotriazole (HOBt) 2.0eq
  • DIEA 4.0eq
  • the Fmoc protective group was removed again with 20% piperidine (PIP) in DMF solution for 30 minutes, and the synthesized L1 (1.5eq) and the condensing agent O-benzotriazole-tetramethylurea hexafluorophosphate ( HBTU) (2.0eq), 1-hydroxybenzotriazole (HOBt) (2.0eq) and DIEA (4.0eq) were dissolved in DMF and added to the solid phase synthesizer, reacted overnight at room temperature, and eluted with DMF 6 times, Lys-(L1) 3 is obtained .
  • PIP piperidine
  • the Fmoc protective group was removed again with 20% piperidine (PIP) in DMF solution for 30 minutes, and the synthesized L1 (1.5eq) and the condensing agent O-benzotriazole-tetramethylurea hexafluorophosphate ( HBTU) (2.0eq), 1-hydroxybenzotriazole (HOBt) (2.0eq) and DIEA (4.0eq) were dissolved in DMF and added to the solid phase synthesizer, reacted overnight at room temperature, and eluted with DMF 6 times to get Lys-(L1) 4 .
  • PIP piperidine
  • the Fmoc protective group was removed again with 20% piperidine (PIP) in DMF solution for 30 minutes, and the synthesized L1 (1.5eq) and the condensing agent O-benzotriazole-tetramethylurea hexafluorophosphate ( HBTU) (2.0eq), 1-hydroxybenzotriazole (HOBt) (2.0eq) and DIEA (4.0eq) were dissolved in DMF and added to the solid phase synthesizer, reacted overnight at room temperature, and eluted with DMF 6 times to get Lys-(L1) 5 .
  • PIP piperidine
  • Lys-L1, Lys-(L1) 2 , Lys-(L1) 3 , Lys-(L1) 4 , and Lys-(L1) 5 were eluted three times with dimethyl sulfoxide (DMSO), respectively.
  • DMSO dimethyl sulfoxide
  • EDC 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Fmoc-Glu-OtBu 1.5eq
  • condensing agent O -Benzotriazole-tetramethylurea hexafluorophosphate HBTU
  • 1-hydroxybenzotriazole HBt
  • DIEA 4.0eq
  • the freshly prepared reaction solution containing FA-NHS was directly added to the solid phase synthesizer coupled with Lys-(L1) 5- Glu, and added DIEA (4.0eq), and reacted overnight in the dark at room temperature.
  • DIEA difluoroacetic acid
  • Tris triisopropylsilane
  • the freshly prepared reaction solution containing FA-NHS was directly added to the solid phase synthesizer coupled with Lys-(L2) 3 -Glu, and added DIEA (4.0eq), and reacted overnight in the dark at room temperature.
  • DIEA 4.0eq
  • After washing 6 times with DMSO, and eluting with DCM 6 times, add 8.0 mL of eluent (trifluoroacetic acid (TFA)/triisopropylsilane (Tris)/water 95:2.5:2.5) and wash at room temperature Take off for 1h.
  • TFA trifluoroacetic acid
  • Tris triisopropylsilane
  • Lys-L1-FA Respectively add Lys-L1-FA, Lys-(L1) 2 -FA, Lys-(L1) 3 -FA, Lys-(L1) 4 -FA, Lys-(L1) 5 -FA, Lys-(L1) 3 -Glu-FA, Lys-(L1) 5 -Glu-FA, Lys-(L2) 3 -Glu-FA or Lys-(L1) 3 -Asp-FA(1.0eq) dissolved in DMSO, then add DIEA( 5.0eq) and Pyro-NHS (0.9eq), and react overnight in the dark at room temperature.
  • Pyro-Lys-(L1) 3 -Asp-FA High resolution mass spectrometry (MALDI-TOF) charge-to-mass ratio C 104 H 146 N 20 O 30 2+ for [M/2+H] + , theoretical molecular weight 1078.5257, we found 1078.5294.
  • MALDI-TOF High resolution mass spectrometry
  • the recording scan range of the fluorescence emission spectrum is from 600nm to 800nm, and the excitation wavelength is 680nm.
  • the recording scan range of the fluorescence excitation spectrum is from 450nm to 670nm, and the emission wavelength is 668nm.
  • the samples were all prepared into DMSO solution with a concentration of 2.0 ⁇ M.
  • the slit widths for excitation and emission are 1 nm and 2 nm, respectively.
  • the UV absorption spectra of Pyro and Pyro-Lys-(L1) 3 -Glu-FA were measured using the American Cary 5000 UV spectrophotometer.
  • the samples were prepared into a solution with a concentration of 10 ⁇ M, the solvents were DMSO, phosphate buffered saline (PBS) containing 0.05% Tween-80, phosphate buffered saline (PBS) containing 1% polyoxyethylene castor oil (CEL) and PBS Solution (the content of DMSO in various solvents is 0.1%).
  • the scanning wavelength ranges from 300nm to 800nm, the resolution is 1nm, and the scanning rate is 600nm per minute.
  • DPBF singlet oxygen quencher 1,3-diphenylisobenzofuran
  • the scanning wavelength range is from 350nm to 800nm with a resolution of 1nm. Scan The rate is 600nm per minute.
  • a linear fit was performed to the irradiation time by the reduced absorbance value of DPBF at 417 nm, as shown in Figure 7B.
  • ⁇ Std ⁇ is the singlet oxygen quantum yield of the reference compound Pyro in DMSO
  • ⁇ Std ⁇ 0.52
  • Rs.m.DPBF and RStd DPBF are the absorbance values of the test compound and the reference compound at 417nm that decrease with the irradiation time.
  • the linear slope, AStd 680 and As.m.680 are the absorbance values of the test compound and the reference compound at 680 nm, respectively.
  • all tested compounds have high singlet oxygen quantum yields in DMSO, especially the compound Pyro-Lys-(L1) 3 -Glu-FA has the strongest ability to generate singlet oxygen.
  • the singlet oxygen quantum yield data in the aqueous solution (the DMSO content is 0.1%) is used as a reference.
  • ABDA 9,10-anthracenyl-bis(methylene)dimalonic acid, 50nM was used as an indicator for singlet oxygen measurement in aqueous solution.
  • the decay rate at 400nm was recorded under different illumination durations, and the singlet oxygen quantum yield in aqueous solution was obtained according to the following formula:
  • K RB and K PS are the decomposition rate constants of the reference compound (RB) and the test compound in PBS solution containing 1% CEL, PBS solution containing 0.05% Tween-80, and PBS solution, respectively, where RB refers to Rose Bengal, whose UV absorption data measured in an aqueous solution are used as control data).
  • a RB and A PS represent the light absorbed by the reference compound (RB) and the test compound, respectively, which are determined by integrating the absorption band in the wavelength range of 400-700 nm.
  • [Phi] RB is an RB singlet oxygen quantum yield in aqueous solution is 0.75.
  • the compound Pyro has a high singlet oxygen quantum yield in a PBS solution containing 1% CEL and a PBS solution containing 0.05% Tween-80, but due to the poor water solubility of Pyro, it is The singlet oxygen quantum yield in pure PBS solution is very low; while the compound Pyro-Lys-(L1) 3 -Glu-FA has a higher singlet oxygen quantum yield in all solutions (see Figure 8D) .
  • the singlet oxygen quantum yield is the most important factor affecting cell lethality in photodynamic therapy, so increasing the singlet oxygen quantum yield in physiological solution is very helpful for photodynamic therapy.
  • the DMSO content in the dilutions of each compound is less than one thousandth.
  • KB cells human oral epidermal cancer cell line
  • A431 cells human skin cancer cell line
  • NCI-N87 cells human gastric cancer cell line
  • A549 cells human lung cancer cell line
  • LO2 cells human Normal liver cell line
  • the cell culture plate was taken out, and a light radiation dose of 40 mW/cm 2 was given for 10 minutes at a distance of about 2 cm from the culture plate with a 660 nm light source. Then, put the 96-well cell culture plate back into the cell incubator and continue to culture overnight. Prepare 0.5mg/ml MTT solution with fresh medium.
  • the IC 50 value of Pyro-FA is about 500 nM in the study of the drug activity at the KB cell level; in the Chinese patent application No. 201710550348.1, the most preferred compound Pyro was selected.
  • the IC 50 value of PEG1K-FA to positive KB cells is about 136nM.
  • Table 2 when the introduction of L1, Pyro-Lys-L1- FA IC 50 value 53.32nM, Pyro-Lys- (L1) 2 -FA IC 50 value 23.15nM.
  • the half inhibitory concentration (IC 50 value) of Pyro-Lys-(L1) 3 -Glu-FA of the present application on positive KB cells is reduced by more Hundred-fold, that is, the lethality of positive cell lines is increased by hundreds of times.
  • KB cells with high folate receptor expression and A549 cells with low folate receptor expression in log phase were taken.
  • Pyro-Lys-(L1) 3 -Glu-FA and Temoporfin and Pyro (as a control) were respectively formulated into 10mM DMSO stock solution, and then diluted with the corresponding complete medium to different drug concentrations (0.1nM, 1nM, 10nM, 20nM and 100nM) dilutions, in the cell culture incubator for 4 hours in the dark.
  • KB cells were plated in a 6-well plate at a density of 5 ⁇ 10 4 cells/well and cultured in a carbon dioxide incubator for 16 hours.
  • 3- Glu-FA and Temoporfin were incubated in a fresh medium for 4 hours in a cell culture incubator, and then placed under a 660nm light source and irradiated for 10 minutes.
  • the cells were digested with trypsin, and centrifuged in a flow tube at 1000rpm/min at 4°C for 3min. For each flow tube, resuspend the cells with 100 ⁇ L of 1 ⁇ binding buffer, add 1 ⁇ L PI working solution and 4 ⁇ L YF488-Annexin V, respectively, and incubate on ice for 15 min in the dark. Then, 400 ⁇ L of 1 ⁇ binding buffer was added to each tube again, and apoptosis was detected by BD FACSCalibur flow cytometer.
  • YF488-Annexin V is excited with a 488nm laser, the fluorescence emission spectrum of the detection drug is at 530nm (FITC channel), and the emission spectrum of PI channel is about 617nm.
  • folate molecules can significantly inhibit the killing ability of Pyro-Lys-(L1) 3 -Glu-FA on KB cells, while the killing ability of Pyro and Temoporfin on cells is not affected by the additional folate molecules. Impact. These experimental results confirmed that under the condition of giving light radiation, Pyro-Lys-(L1) 3 -Glu-FA enters the cell by binding to the folate receptor and through receptor-mediated endocytosis, thereby exerting its photodynamic force active.
  • KB cells, A431 cells, NCI-N87 cells, A549 cells, and LO2 cells were plated in a 96-well plate at a density of 1 ⁇ 10 4 cells/well, and cultured in an incubator for 16 hours.
  • Add corresponding concentrations of Pyro, Pyro-Lys-(L1) 3 -Glu-FA or Temoporfin into the culture wells, and place the culture plate in the cell culture incubator for 4 hours, and then place it under a light source of 660nm. Irradiate with a light intensity of 40mW/cm 2 for 10 minutes, and then continue to culture overnight in a cell incubator.
  • Dark toxicity refers to the toxicity of the photosensitizer to cells in the absence of light radiation to excite the photosensitizer. As an ideal photosensitizer, it should have no obvious non-exciting dark toxicity.
  • Pyro-Lys- (L1) 3 -Glu -FA IC 50 on KB cell line is 1.403nM
  • on the IC A431 cell line 50 is 103.3nM
  • Pyro-Lys- (L1) 3 -Glu -FA on IC A549 cell line 50 is 2864nM
  • the killing activity of Pyro-Lys-(L1) 3 -Glu-FA on the KB cell line is 2000 times that of the A549 cell line and 1660 times that of the LO2 cell line.
  • the IC 50 values of Pyro and Temoporfin for different cell lines in the control group are between 65-80 nM and 100-200 nM, with only a slight difference. This slight difference comes from the difference between Pyro and Temoporfin. The sensitivity of moporfin is different.
  • the introduction of the targeting ligand-folic acid enables Pyro-Lys-(L1) 3 -Glu-FA to selectively kill cell lines with different expression levels of folate receptors, which is beneficial to reduce the effect of photosensitive drugs on normal tissues.
  • the killing effect is beneficial to reduce the effect of photosensitive drugs on normal tissues.
  • Pyro-Lys-(L1) 3 -Glu-FA showed no obvious cytotoxicity to different cell lines without light, which proves that Pyro-Lys-(L1) 3 -Glu- FA meets the important properties of ideal photosensitizers without toxic and side effects in the absence of light.
  • the mouse subcutaneous tumor models are KB tumor model, A431 tumor model and NCI-N87 tumor model.
  • Female BALB/c nude mice aged 6 to 8 weeks were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., and have been kept in an SPF environment.
  • the specific experimental method is as follows: First, calculate the number of tumor cells required according to the number of mice, and culture the corresponding tumor cells in a reasonable amount. In each tumor model in this experiment, each mouse requires 3 ⁇ 10 6 cells. After the cultured cells occupy about 80% of the space of the bottom plate of the petri dish, the cells are digested in a manner similar to passage, and centrifuged at 1000 rpm for 3 minutes in a centrifuge pre-cooled to 4°C.
  • Subcutaneous tumor inoculation method for mice After anesthetizing the mouse with 15% isoflurane, wipe and disinfect the right side of the mouse with 75% alcohol, and then use a syringe to draw 100 ⁇ L (3 ⁇ 10 6 cells) tumor cell suspension , And inject the cells under the skin of the mouse, then turn the needle tip upside down, pull out the mouse, and mark it. After 5 days, the tumor volume of the mouse was detected every 2 days, and the tumor volume and the weight change of the mouse were recorded.
  • -Lys-(L1) 5 -Glu-FA the content of DMSO administered intravenously to each mouse is 2.5%), and starting from the injection time point, use the in vivo imaging system to scan 0.5h, 1h, 2h, The distribution of drugs at 3h, 4h and 6h.
  • the compound can be quickly concentrated in the liver and kidney of mice 1 hour after administration
  • the drug is gradually metabolized at the site of the tumor and tumor tissues, and over time, the drug is gradually metabolized; 2 hours after administration, the content of the compound in the liver is significantly reduced, and the compound is mainly concentrated in the tumor site.
  • mice The above method was used to construct a mouse subcutaneous tumor KB tumor model (5 mice per group).
  • the same dose (50 nmol) of Pyro-Lys-(L2) 3 -Glu-FA or Pyro-Lys-(L1) 3 -Asp-FA each The DMSO content of intravenous administration in mice is less than 2.5%), and the injection time point is used as the starting point, and the in vivo imaging system is used to scan the distribution of the drug in the mice at 1h, 2h, 4h, 6h, 9h and 24h after the tail vein administration happensing.
  • mice A variety of tumor-bearing female BALB/c nude mice were used to study the drug metabolism characteristics of Pyro-Lys-(L1) 3 -Glu-FA, and Pyro and Temoporfin were used as controls.
  • KB cells or A431 cells or NCI-N87 cells were inoculated on the back of mice.
  • the tumor volume reached about 200 mm 3
  • untreated mice were scanned according to the above method as a background control.
  • 50nmol of Pyro, Pyro-Lys-(L1) 3 -Glu-FA or Temoporfin (the content of DMSO administered intravenously per mouse is less than 2.5%) was injected into each group of mice through the tail vein, and The injection time point was used as the starting point.
  • mice For Pyro and Pyro-Lys-(L1) 3 -Glu-FA mice, scan the drug distribution in mice at 0.5h, 1h, 2h, 3h, 4h and 6h after administration, while for Temoporfin Group mice, scan the drug distribution in mice 0.5h, 1h, 2h, 3h, 4h, 6h, 12h, 1d, 2d, 3d, 4d, 6d and 8d after administration.
  • the mice were sacrificed, the organs, tumor tissues and muscle tissues of each group of mice were taken out, and the organs, tumor tissues and muscle tissues of each group of mice were scanned by the in vivo imager.
  • the amount of compound Using the software of the in vivo imaging system, calculate the average fluorescence intensity of the tumor site and the total average fluorescence intensity of each tissue, and calculate the ratio of the two. This ratio can reflect the average enrichment degree of the photosensitive drug in the tumor and the decrease of the photosensitive drug Application potential in skin toxicity.
  • the unit of ROI region of interest is expressed as: [p/sec/cm 2 /sr]/[ ⁇ W/cm 2 ].
  • the distribution characteristics of Pyro, Pyro-Lys-(L1) 3 -Glu-FA and Temoporfin in A431 and NCI-N87 tumor model mice are similar to those in KB tumor model mice.
  • Pyro-Lys-(L1) 3 -Glu-FA can be observed to be significantly enriched in the tumor tissue 2h after administration, while Pyro has been concentrated in liver tissue in large numbers and has no obvious accumulation in tumor tissue , And the accumulation of temoporfin in the tumor tissue reaches the maximum at 12h-24h after administration, and the metabolism time in the body is still as long as 8d.
  • mice of each tumor model were sacrificed 24h after the administration, and the heart, liver, spleen, lung, kidney, tumor and muscle tissues of each group of mice were taken out, and directly compared and observed in the animal live imaging instrument .
  • Pyro-Lys-(L1) 3 -Glu-FA was enriched in KB, A431 and NCI-N87 tumor tissues and kidney tissues, while the content in liver tissues was higher. less.
  • the vast majority of Pyro accumulates in liver tissue.
  • Temoporfin also accumulates in tumor tissues, it also accumulates in liver, lung, kidney and other tissues.
  • KB, A431, NCI-N87 tumor models with different expression of folate receptor were used to study the therapeutic effect of Pyro-Lys-(L1) 3 -Glu-FA.
  • 3 ⁇ 10 6 KB cells were subcutaneously inoculated on the right side of the back of the mouse. After about 18 days, the tumor volume of the mice reached about 200 mm 3 .
  • the KB transplanted tumor mice were randomly divided into the following 5 groups: (1) 200 ⁇ L PBS/light+(light) (control group); (2) 60nmol Pyro/light- (no light); (3) 60nmol Pyro/light+ (Lighting); (4) 60nmol Pyro-Lys-(L1) 3 -Glu-FA/light-(no light); and (5) 60nmol Pyro-Lys-(L1) 3 -Glu-FA/light+(lighting ), 5 mice per group (the content of DMSO administered intravenously to each mouse is 3%).
  • the light energy given to each light group was 120J/cm 2 (200mW/cm 2 , 10min). 2h after the tail vein administration was used as the irradiation time point of photodynamic therapy for mice. Every two days, a vernier caliper was used to record the volume change of the mouse tumor and the weight change of the mouse. When the tumor volume of the mouse reached 1500 mm 3 , the mouse was considered dead and the recording was stopped, and the mouse was continuously observed for 95 days.
  • mice Use the above method to inoculate 3 ⁇ 10 6 A431 or NCI-N87 tumor cells subcutaneously in mice respectively, and randomly divide the mice into the following three groups (5 in each group): (1) PBS/light + (light); (2) 60nmol Pyro/light+(light); and (3) 60nmol Pyro-Lys-(L1) 3- Glu-FA/light+(light).
  • PBS/light + (light) The light was irradiated to the tumor tissue site at 200mW/cm 2 for 10 minutes, and the tumor volume and weight change of the mice were monitored every two days.
  • mice in the Pyro-Lys-(L1) 3 -Glu-FA/light+ group developed inflammatory edema near the tumor, and the edema was relieved after 2 days
  • Pyro- In the Lys-(L1) 3 -Glu-FA/light+ group mice in the Lys-(L1) 3 -Glu-FA/light+ group had scabs at the tumor site and the tumor volume began to shrink. Two weeks later, the scab fell off completely and the tumor disappeared. No recurrence was observed after 95 days of continuous observation.
  • the inflammation at the tumor site was mild, the edema almost disappeared after 2 days, there was no scab phenomenon, and the tumor volume was suppressed in a short time; but the tumor volume increased rapidly after 10 days.
  • the mice in the PBS/light+ group showed no edema after light exposure, and the tumor volume was comparable to that in the non-light group (Pyro/light-group and Pyro-Lys-(L1) 3 -Glu-FA/light-group).
  • the tumor volume of mice with increased rapidly, and the tumor volume quickly reached 1500 mm 3 about 18 days after light or administration.
  • the experimental group without light or photosensitizer there was no inhibitory effect on tumor volume growth.
  • the A431 and NCI-N87 transplanted tumor mice were treated with photodynamic therapy using the above methods.
  • mice in the Pyro-Lys-(L1) 3 -Glu-FA/light+ group also showed edema after light exposure, and the tumor volume of the mice gradually decreased, but within 15-25 days after the first photodynamic therapy , The tumors of some mice grew again.
  • the recurring tumor grows to about 100 mm 3
  • the recurrent tumors are treated with a second administration (also 60 nmol is administered to the tail vein, and 200 J/cm 2 of light energy is given 2 hours after administration).
  • the tumor showed no signs of recurrence.
  • mice By monitoring the weight change of the mice, the effect of the test compound on the physiological state of the mice during the photodynamic therapy is indirectly monitored.
  • the body weight of the Pyro-Lys-(L1) 3 -Glu-FA/light+ group All of them showed a tendency to decrease 10 days after the first treatment, but then their body weight slowly increased until it was the same as the body weight of the other groups of mice, which stabilized and showed a slow upward trend.
  • the survival rate of mice in this group can reach 100% after one or two photodynamic treatments.
  • Pyro-Lys-(L1) 3 -Glu-FA is a highly effective photosensitizer with low toxic and side effects that can be used for anti-tumor photodynamic therapy.
  • mice weighing 18-20g were fasted for 6 hours before administration, and then divided into three groups (5 mice in each group): (1) 5 ⁇ L DMSO+1 ⁇ L castor oil, protected from light; (2) ) 500nmol Pyro+1 ⁇ L castor oil, protected from light; and (3) 500nmol Pyro-Lys-(L1) 3 -Glu-FA, protected from light.
  • the drug was administered every 2 days for a total of 4 times.
  • the weight changes of the mice in each group were detected every day, and the physiological reactions and behaviors of the mice were monitored.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

公开了通式(I)的光敏剂、其制备方法及其在肿瘤光动力治疗和肿瘤成像诊断中的应用。在所述光敏剂中，作为肿瘤靶向配基的叶酸通过任选地具有酸性氨基酸片段的不同链长的连接部分与卟吩型光敏部分连接。

Description

不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用 技术领域
本申请涉及具有肿瘤靶向性的光敏剂、其制备方法及其在肿瘤光动力治疗和肿瘤成像诊断中的应用。在所述光敏剂中，作为肿瘤靶向配基的叶酸(FA)通过任选地具有酸性氨基酸片段的不同链长的连接部分与作为光敏部分(PS)的卟吩类化合物连接。
背景技术
目前，临床上治疗癌症的方法主要包括放疗、化疗和手术治疗三种手段。然而，这些传统的治疗方法在杀伤肿瘤组织的同时也会损伤机体正常的组织，造成全身毒性。光动力治疗(Photodynamic therapy,PDT)是新型的局部的低毒的肿瘤治疗方案。光动力治疗所使用的光敏剂具有较低的暗毒性，并且光动力治疗的直接作用位点是光辐射部位的病变组织，用一定的激发波长照射疾病部位，光敏剂才会被激活从而产生杀伤能力。光敏剂的光学性质决定了其在肿瘤组织部位富集后，可以产生明显的荧光信号，因此，可以将诊断和治疗一体化并为临床手术过程提供指导。
目前的光敏剂主要为卟吩类化合物。卟吩类化合物的吸收波长为约680nm，例如：焦脱镁叶绿酸a(Pyro)、甲基嗜焦素烷基醚衍生物(HPPH)、二氢卟吩e6(Ce6)、维替泊芬(BPD-MA，vertoporfin)和替莫泊芬(Temoporfin，商品名：Foscan)等。焦脱镁叶绿酸a(Pyropheophorbide a,Pyro)在波长668nm下的消光系数为3.79×10 4L mol -1cm -1，并具有较强的单线态氧量子产率(0.52)，因此具有较强的细胞光动力杀伤活性。但是，Pyro的水溶性差，并且基本上不具有肿瘤定位能力，在肿瘤组织中的浓度低，而在正常组织中的浓度高，在光动力治疗过程中会对正常组织产生较为严重的光毒副作用。因此，通过对Pyro进行结构改造，解决限制其临床应用的缺点，有望开发出所需的光敏剂。
提高光敏剂在肿瘤部位的富集能力，从而能有效增强肿瘤光动力治疗的疗效。通过将光敏剂与在肿瘤表面高表达受体的靶向配基偶联，可赋予光敏剂主动靶向肿瘤的能力，这成为制备肿瘤特异性光敏剂的有效策略。叶酸受体普遍高表达于多数肿瘤细胞表面，并且小分子叶酸可以作为理想的肿瘤靶向配基而被广泛应用于肿瘤成像和治疗领域中。
在第201610628964.X号中国专利申请中，通过将作为肿瘤靶向基团的叶酸经由半胱氨酸与Pyro相连，提高Pyro的肿瘤富集能力。所制备的化合物(Pyro-FA)对叶酸高表达的皮下肿瘤表现出良好的选择能力和非常有效的光动力治疗效果。
在第201710550348.1号中国专利申请中，通过向将小分子靶向配基叶酸与光敏剂Pyro连接的含有半胱氨酸的连接链中引入高度亲水的不同链长的聚乙二醇，改善了Pyro-FA的性能。合成后的化合物具有良好的水溶性，并增强了对肿瘤的靶向富集能力，改善了光敏剂在体内的代谢分布特性，增强了动物水平上的光动力治疗的效果。
虽然通过聚乙二醇连接Pyro与叶酸分子可以提高光敏剂的水溶性，但是仍然存在一些问题。例如，用于PEG化的作为连接链的聚乙二醇的分子量为平均分子量，分子结构不固定，分子量也不固定，化合物纯化比较困难，而且依然不能确定能够使得光敏药物分子具有优异的肿瘤特异富集功能和高效的光敏活化杀能力的具体结构。因此，仍然亟需具有优异的肿瘤靶向性和改善的体内代谢分布特性的光敏剂。
发明概述
一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐：
A-Lys-(L) m-(B) o-C
(I)
其中
A和C独立地选自叶酸基团(FA)和卟吩型光敏部分(PS)，并且当A和C中的任一个为叶酸基团时，则另一个为卟吩型光敏部分；
Lys表示
Figure PCTCN2020094308-appb-000001
L独立地选自L1或L2；
Figure PCTCN2020094308-appb-000002
B表示酸性氨基酸片段；
m为1-5的整数；
o为0或1；以及
p为4-8的整数。
另一方面，本申请提供了制备通式(I)的光敏剂的方法，其包括：
1)合成连接子L；
2)使m个连接子L与Lys连接以获得片段Lys-(L) m；
3)任选地使片段Lys-(L) m与酸性氨基酸片段B连接以获得片段Lys-(L) m-(B) o；
4)将片段Lys-(L) m-(B) o与叶酸FA连接以获得片段Lys-(L) m-(B) o-FA；以及
5)将片段Lys-(L) m-(B) o-FA与卟吩型光敏化合物PS连接以获得通式(I)的光敏剂；或者
6)将片段Lys-(L) m-(B) o与卟吩型光敏化合物PS连接以获得片段Lys-(L) m-(B) o-PS；以及
7)将片段Lys-(L) m-(B) o-PS与叶酸FA连接以获得通式(I)的光敏剂；
其中Lys、L、m、B、o、FA和PS如上文所定义。
另一方面，本申请提供了包含通式(I)的光敏剂或其药学上可接受的盐和药学上可接受的赋形剂的药物组合物。
另一方面，本申请提供了包含通式(I)的光敏剂或其药学上可接受的盐的成像诊断剂。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备用于治疗肿瘤的药物中的用途。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备肿瘤成像诊断剂中的用途。
还一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备通过光动力疗法来治疗肿瘤的药物中的用途。
再一方面，本申请提供了治疗肿瘤的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
再一方面，本申请提供了使肿瘤成像以进行诊断的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
还一方面，本申请提供了通过光动力疗法来治疗肿瘤的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
再一方面，本申请提供了用于治疗肿瘤的通式(I)的光敏剂或其药学上可接受的盐或其药物组合物。
再一方面，本申请提供了用于使肿瘤成像以进行诊断的通式(I)的光敏剂或其药学上可接受的盐或其药物组合物。
还一方面，本申请提供了通过光动力疗法来治疗肿瘤的通式(I)的光敏剂或其药学上可接受的盐或其药物组合物。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在治疗肿瘤中的用途。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在使肿瘤成像以进行诊断中的用途。
还一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在通过光动力疗法来治疗肿瘤中的用途。
附图说明
图1是L1的合成方法示意图。
图2是Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)的合成方法示意图。
图3是Pyro-Lys-(L1) m-Glu-FA(m＝3或5)的合成方法示意图。
图4是Pyro-Lys-(L2) 3-Glu-FA的合成方法示意图。
图5是Pyro-Lys-(L1) 3-Asp-FA的合成方法示意图。
图6是Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA的高效液相色谱分析图。
图7A和图7B分别是Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA的荧光光谱图以及它们在DMSO中的单线态氧量子产率测定图。
图8A至图8D是Pyro-Lys-(L1) 3-Glu-FA和Pyro在不同溶液中的紫外吸收光谱图及在PBS溶液中的单线态氧量子产率测定图。
图9显示了Pyro、Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA在体外KB细胞水平上的生物活性。
图10显示了Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别在KB和A549细胞中的选择性内吞。
图11显示了Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别与叶酸对KB细胞系活性的竞争性抑制。
图12显示了Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬在不同细胞系水平上的生物活性和暗毒性。
图13显示了Pyro、Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA在KB移植瘤小鼠体内随时间分布的药物代谢过程。
图14显示了Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA在KB移植瘤小鼠 体内随时间分布的药物代谢过程。
图15A至图15E显示了Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别在KB、A431和NCI-N87移植瘤小鼠体内随时间分布的药物代谢过程及它们在各器官组织、肿瘤组织和肿瘤附近肌肉组织的富集(Ht：心；Lv：肝；Sp：脾；Lu：肺；Kn：肾；Tm：肿瘤；Ms；肌肉)。
图16A至图16C分别显示了Pyro和Pyro-Lys-(L1) 3-Glu-FA分别对KB、A431和NCI-N87移植瘤小鼠的肿瘤光动力治疗效果。
图17显示了Pyro-Lys-(L1) 3-Glu-FA和Pyro对正常小鼠的急性毒性。
发明详述
一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐：
A-Lys-(L) m-(B) o-C
(I)
其中
A和C独立地选自叶酸基团(FA)和卟吩型光敏部分(PS)，并且当A和C中的任一个为叶酸基团时，则另一个为卟吩型光敏部分；
Lys表示
Figure PCTCN2020094308-appb-000003
L表示连接子(linker)，其独立地选自L1或L2；
Figure PCTCN2020094308-appb-000004
B表示酸性氨基酸片段；
m为1-5的整数；
o为0或1；以及
p为4-8的整数。
在本申请的实施方案中，所述叶酸基团优选地具有如下结构：
Figure PCTCN2020094308-appb-000005
在本申请的实施方案中，所述卟吩型光敏部分独立地选自Pyro的基团、Ce6的基团、HPPH的基团和Vertoporfin的基团，优选为Pyro的基团或Ce6的基团，更优选为Pyro的基团。
Figure PCTCN2020094308-appb-000006
在本申请的优选实施方案中，所述Pyro的基团具有结构
Figure PCTCN2020094308-appb-000007
在本申请的实施方案中，所述酸性氨基酸片段为谷氨酸片段和天冬氨酸片段；优选地，所述谷氨酸片段具有结构
Figure PCTCN2020094308-appb-000008
以及所述天冬氨酸片段具有结构
Figure PCTCN2020094308-appb-000009
在本申请的实施方案中，m为1-5的整数；更优选为2、3、4或5，还更优选为3或5；最优选为3。在本申请的实施方案中，o优选为1。在本申请的实施方案中，L2中的重复单元-CH 2CH 2O-的个数p还可以为4、5、6、7或8，优选为6，即，L2具有结构
Figure PCTCN2020094308-appb-000010
在本申请的优选实施方案中，-(L) m-具有如下结构之一：
Figure PCTCN2020094308-appb-000011
在本申请的更优选实施方案中，-(L) m-(B) o-具有如下结构之一：
Figure PCTCN2020094308-appb-000012
在本申请的实施方案中，通式(I)的光敏剂优选地选自如下化合物之一或其药学上可接受的盐：
Pyro-Lys-L1-FA：
Figure PCTCN2020094308-appb-000013
Pyro-Lys-(L1) 2-FA：
Figure PCTCN2020094308-appb-000014
Pyro-Lys-(L1) 3-FA：
Figure PCTCN2020094308-appb-000015
Pyro-Lys-(L1) 4-FA：
Figure PCTCN2020094308-appb-000016
Pyro-Lys-(L1) 5-FA：
Figure PCTCN2020094308-appb-000017
Pyro-Lys-(L1) 5-Glu-FA：
Figure PCTCN2020094308-appb-000018
Pyro-Lys-(L1) 3-Glu-FA：
Figure PCTCN2020094308-appb-000019
Pyro-Lys-(L2) 3-Glu-FA：
Figure PCTCN2020094308-appb-000020
Pyro-Lys-(L1) 3-Asp-FA：
Figure PCTCN2020094308-appb-000021
另一方面，所述通式(I)的光敏剂的药学上可接受的盐包括药学上可接受的酸加成盐或药学上可接受的碱加成盐，其分别通过使通式(I)的光敏剂与药学上可接受的无机酸或有机酸或者与药学上可接受的无机碱或有机碱反应来获得。
另一方面，本申请提供了制备通式(I)的光敏剂的方法，其包括：
1)合成连接子L；
2)使m个连接子L与Lys连接以获得片段Lys-(L) m；
3)任选地使片段Lys-(L) m与酸性氨基酸片段B连接以获得片段Lys-(L) m-(B) o；
4)将片段Lys-(L) m-(B) o与叶酸FA连接以获得片段Lys-(L) m-(B) o-FA；以及
5)将片段Lys-(L) m-(B) o-FA与卟吩型光敏化合物PS连接以获得通式(I)的光敏剂；或者
6)将片段Lys-(L) m-(B) o与卟吩型光敏化合物连接以获得片段Lys-(L) m-(B) o-PS；以及
7)将片段Lys-(L) m-(B) o-PS与叶酸FA连接以获得通式(I)的光敏剂；
其中Lys、L、m、B、o、FA和PS如上文所定义。
在本申请的实施方案中，连接子L独立地选自如上文所定义的L1或L2，其中L1可以通过下文所描述的合成方法获得，L2可以通过下文所描述的合成方法获得或者可以商购。
Figure PCTCN2020094308-appb-000022
连接子L1的合成方法
在本申请的实施方案中，步骤2)至步骤5)的合成路线如下所示，其中以3个连接子L1作为-(L) m-的实例，以谷氨酸片段作为酸性氨基酸片段的实例，以Pyro作为卟吩型光敏化合物的实例：
Figure PCTCN2020094308-appb-000023
通过固相合成和液相合成相结合的方式，以非常简单实用的方式得到了一系列的通式(I)的光敏剂。利用二氯三苯甲基树脂采用Fmoc固相合成多肽的方法合成了具有 Lys基团和m个连接子L及任选地具有酸性氨基酸片段的不同链长的连接部分，再与FA配基偶联，最后通过与诸如Pyro的活性酯等卟吩型光敏化合物PS的活性酯进行缩合得到产物(即，通式(I)的光敏剂)，例如Pyro-Lys-L1-FA、Pyro-Lys-(L1) 2-FA、Pyro-Lys-(L1) 3-FA、Pyro-Lys-(L1) 4-FA、Pyro-Lys-(L1) 5-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA。所述产物用HPLC进行纯度分析，并用HRMS进行分子量表征。
在本申请的实施方案中，本申请提供上述光敏剂的制备方法，其详细的合成路线如图1-图5所示。
另一方面，本申请提供了包含通式(I)的光敏剂或其药学上可接受的盐和药学上可接受的赋形剂的药物组合物。
术语“药学上可接受的赋形剂”包括任何和所有的溶剂、分散介质、抗菌剂、防腐剂、等渗剂、无热原水、生理盐水和磷酸盐缓冲溶液等。这些介质和试剂用于药物活性物质的用途是本领域熟知的。除非任何常规介质或试剂与活性成分不相容，否则考虑其在治疗组合物中的用途。另外，可以包括各种辅料，例如本领域常用的辅料。这些和其它此类化合物描述于文献中，例如Merck Index,Merck&Company,Rahway,NJ。例如，Gilman et al.(Eds.)(1990)；Goodman and Gilman’s:The Pharmacological Basis of Therapeutics,8th Ed.,Pergamon Press中描述了在药物组合物中包含各种组分的考虑因素。
本申请公开的通式(I)的光敏剂或其药学上可接受的盐的给药可以通过用于具有类似功用的药剂的任何可接受的给药方式实施，包括但不限于口服给药和注射给药，例如皮下、静脉内、局部、透皮、腹膜内和肌内给药等。口服给药和肠胃外给药通常用于治疗作为优选实施方案的主体的适应症。
可以将如上所述的通式(I)的光敏剂或其药学上可接受的盐配制成药物组合物以用于治疗肿瘤。使用标准的药物制剂技术，例如Remington's The Science and Practice of Pharmacy,21st Ed.,Lippincott Williams&Wilkins(2005)中公开的那些，该文献通过引用以其整体并入本文。
另一方面，本申请提供了包含通式(I)的光敏剂或其药学上可接受的盐的成像诊断剂。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备用于治疗肿瘤的药物中的用途。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备肿瘤成像诊断剂中的用途。
还一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在制备通过光动力疗法来治疗肿瘤的药物中的用途。
再一方面，本申请提供了治疗肿瘤的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
再一方面，本申请提供了使肿瘤成像以进行诊断的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
还一方面，本申请提供了通过光动力疗法来治疗肿瘤的方法，其包括将通式(I)的光敏剂或其药学上可接受的盐或其药物组合物向有需要的个体给药。
再一方面，本申请提供了用于治疗肿瘤的通式(I)的光敏剂或其药学上可接受的盐或其药物组合物。
再一方面，本申请提供了用于使肿瘤成像以进行诊断的通式(I)的光敏剂或其药学上可接受的盐或其药物组合物。
还一方面，本申请提供了通过光动力疗法来治疗肿瘤的通式(I)的光敏剂或其药学 上可接受的盐或其药物组合物。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在治疗肿瘤中的用途。
再一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在使肿瘤成像以进行诊断中的用途。
还一方面，本申请提供了通式(I)的光敏剂或其药学上可接受的盐或其药物组合物在通过光动力疗法来治疗肿瘤中的用途。
在本申请的实施方案中，所述肿瘤为叶酸受体高表达的癌症，包括但不限于口腔表皮样癌、表皮鳞癌和胃癌。
在本申请中，设计并合成了一系列具有不同链长的任选地具有酸性氨基酸片段如Glu片段和Asp片段的连接部分，并通过该连接部分将作为肿瘤靶向配基的叶酸与诸如Pyro的卟吩型光敏部分连接而获得了通式(I)的光敏剂，然后对所述光敏剂进行了光物理性质研究以及在体外和体内的生物活性评价。
本申请的发明人经研究发现，引入亲水性较强的连接部分和更多的羧基官能团能显著提高诸如Pyro的卟吩型光敏化合物的水溶性，以及提高药物分子的水溶性可以提高药物在肿瘤部位的富集能力并降低药物在小鼠体内的背景吸收。
通过在人口腔表皮样癌KB细胞系(叶酸受体高表达)、人类表皮鳞癌A431细胞系(叶酸受体高表达)、人胃癌NCI-N87细胞系(叶酸受体高表达)、A549人肺癌细胞(叶酸受体低表达)和LO2人正常肝细胞系上以及在KB、A431或NCI-N87三种异种移植肿瘤模型小鼠上对通式(I)的光敏剂进行生物学研究和体内药物代谢研究，发现相比于现有光敏剂Pyro和替莫泊酚，本申请的通式(I)的光敏剂至少具有如下优势之一：显示出良好的肿瘤选择性；对叶酸受体高表达的肿瘤表现出优异的肿瘤特异性富集能力和肿瘤细胞杀伤能力；可以实现快速富集和快速清除；可以降低对正常组织的毒副作用。
因此，本申请的通式(I)的光敏剂在肿瘤光动力治疗领域和肿瘤成像诊断领域具有良好的应用前景，具备开发成药物的潜力，特别是具有含3个连接子(优选地，3个连接子的总分子量为约1K)和1个酸性氨基酸片段的连接部分的通式(I)的光敏剂，例如Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA，更具备开发成药物的潜力。
实施例
本领域技术人员会理解，下述实施例仅是用于示例性说明本发明但不意味着限制本发明。实施例中未描述的具体技术或条件可以按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。未注明生产厂商的试剂或仪器均是可以通过市场获得的常规产品。
缩写语
PDT：光动力治疗；Pyro：焦脱镁叶绿酸a；FA：叶酸；Lys：赖氨酸；Glu：谷氨酸；Asp：天冬氨酸；PEG：聚乙二醇；Et 2O：乙醚；THF：四氢呋喃；Et 3N：三乙胺；DIEA：N,N-二异丙基乙胺；EA：乙酸乙酯；DCM：二氯甲烷；EtOH：乙醇；MeOH：甲醇；DMF：N,N-二甲基甲酰胺；DMSO：二甲基亚砜；HCl：盐酸；NaOH：氢氧化钠；NaHCO 3：碳酸氢钠；Na 2SO 4：硫酸钠；MgSO 4：硫酸镁；Pd/C：钯/碳；H 2：氢气；TFA：三氟乙酸；Tris：三异丙基硅烷；PIP：哌啶；Fmoc-OSu：芴甲氧羰酰琥珀酰亚胺；HBTU：O-苯并三氮唑-四甲基脲六氟磷酸酯；HOBt：1-羟基苯并三唑；DCC：二环己基碳二亚胺；DMAP：4-二甲氨基吡啶；EDC：1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐；NHS：N-羟基丁二酰亚胺；Fmoc：芴甲氧羰基；Boc：叔丁氧 羰基；OtBu：叔丁氧基；Cbz-Cl：苄氧羰基氯；HPLC：高效液相色谱；HRMS：高分辨质谱；CEL：聚氧乙烯蓖麻油；Tween-80：失水山梨醇单油酸酯聚氧乙烯醚，也叫吐温80；PBS：磷酸缓冲液；DPBF：1,3-二苯基异苯并呋喃；ABDA：9，10-蒽基-双(亚甲基)二丙二酸；MTT：噻唑蓝；IC 50值：半抑制剂量；KB：人口腔表皮样癌细胞；A431：人表皮癌细胞；NCI-N87：人胃癌细胞；A549：人肺癌细胞；LO2：人正常肝细胞系；SPF：无特定病原体；
实验材料的来源
Pyro购自宁波洞密生物科技有限公司；FA、二甘醇酐、EDC、NHS和TFA购自天津希恩思生化科技有限公司；Glu、Lys、Cbz-Cl、Fmoc-OSu、三苯基氯树脂、HATU和HoBt购自吉尔生化(上海)有限公司；4,7,10-三氧-1,13-十三烷二胺和Tris购自梯希爱(上海)化成工业发展有限公司；DIEA购自北京百灵威科技有限公司；Fmoc-PEG 6-CH 2CH 2COOH购自成都福瑞康生物科技有限公司；Pd/C购自西安凯立化工有限公司；CO 2和H 2购自天津六方工业气体经销有限公司；MTT、YF488-Annexin V和PI试剂盒和异氟烷购自天津百倍生物科技有限公司；以及其它常规的生物试剂与化学试剂均购自天津化学试剂经销公司。
实施例1.连接子L1的合成
第一步：Cbz-苯酚的合成
如图1所示，在室温下称取苯酚(300.0g)并溶于500ml四氢呋喃(THF)中，加入三乙胺(Et 3N)(532.0ml，1.2eq)，然后用液氮降温至-10℃，再滴加Cbz-Cl(495.0ml，1.1eq)。滴加完毕后，自然升温至室温并搅拌反应过夜。旋蒸除去大部分的THF，加水稀释，并用乙酸乙酯(EA)萃取3次，合并有机相，分别用2N的盐酸(HCl)、饱和碳酸氢钠(NaHCO 3)溶液洗涤两次，再用水、饱和食盐水洗涤，经无水硫酸钠(Na 2SO 4)干燥，旋蒸除去EA，得黄色油状化合物1(857.4g)，产物未经纯化直接用于下一步反应。
第二步：化合物2的合成
在反应瓶中称取4,7,10-三氧-1,13-十三烷二胺(200.0g)并溶于乙醇(EtOH)(100.0ml)中，并加热至70℃，将化合物1(242.4g，1.0eq)的EtOH(400.0ml)溶液快速滴加至反应瓶中，滴加完毕后在70℃下反应过夜。旋蒸除去EtOH，加入3N的盐酸(HCl)并充分搅拌后，用EA洗，然后用2N的氢氧化钠(NaOH)溶液调节混合物的pH至pH＝10，再用EA萃取3次，合并有机相，用饱和食盐水洗，无水硫酸镁(MgSO 4)干燥，旋蒸除去EA，得黄色油状化合物2(160.0g)，产物未经纯化直接用于下一步反应。
第三步：化合物3的合成
将化合物2(50.0g)溶于二氯甲烷(DCM)(500.0ml)中，加入二甘醇酐(19.6g，1.2eq)和N,N-二异丙基乙胺(DIEA)(47.0ml，2.0eq)，在室温搅拌过夜。将反应液依次用1N的盐酸(HCl)、水、饱和食盐水洗，无水硫酸镁(MgSO 4)干燥，旋蒸除去DCM，得黄色油状化合物3(55.0g)，产物未经纯化直接用于下一步反应。
第四步：化合物4的合成
将化合物3(55.0g)溶于甲醇(MeOH)(500.0ml)中，加入Pd/C，并用H 2置换空气，然后在室温下搅拌过夜。过滤除掉Pd/C，用旋蒸除去滤液中的甲醇，得黄色油状化合物4(39.0g)，产物未经纯化直接用于下一步反应。 1H NMR(300MHz,DMSO-d 6)δ9.999-10.034(t,1H),8.431-8.555(m,2H),3.863(s,2H),3.754(s,2H),3.342-3.532(m,12H),3.095-3.137(t,2H),2.854-2.903(t,2H),1.743-1.829(m,2H),1.617-1.705(m,2H)。
第五步：L1的合成
将化合物4(12.6g)溶于N,N-二甲基甲酰胺(DMF)(50.0ml)中，并加入芴甲氧羰酰琥珀酰亚胺(Fmoc-OSu)(15.2g，1.2eq)和N,N-二异丙基乙胺(DIEA)(10.0ml，2.0eq)， 在室温下搅拌过夜。加入4N的盐酸(HCl)将反应液的pH调节至pH＝3，再用EA萃取3次，合并有机相，分别用水、饱和食盐水洗，无水硫酸钠(Na 2SO 4)干燥，旋蒸除去EA，得到粗产物，将其经硅胶柱色谱法纯化，得产物L1(7.0g)，收率33.36％。 1H NMR(300MHz,DMSO-d 6)δ7.850-7.875(d,2H),7.644-7.668(d,2H),7.364-7.412(t,2H),7.239-7.327(t,2H),4.268-4.290(d,2H),4.162-4.207(t,1H),4.081(s,2H),3.923(s,2H),3.335-3.490(m,12H),3.101-3.166(m,2H),2.978-3.042(m,2H),1.562-1.677(m,4H)。
实施例2.Pyro-Lys-(L1) m-FA(m＝1、2、3、4、5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA的合成
A.利用固相方法合成Lys-(L1) m-FA(m＝1、2、3、4、5)化合物的通用程序
如图2所示，在合成前1h，用二氯甲烷对载量为1.0mmol/g的三苯基氯树脂(5.0mmol)进行溶胀处理。将Boc-Lys(Fmoc)-OH(1.0g,1.0mmol)和DIEA(680.0μL,4.0mmol)溶解于DCM(10.0mL)中，然后加入到固相合成器中，在室温下反应过夜。用DCM洗6次，然后将配制的封闭液(DCM:MeOH:DIEA＝17:1:2)加入到固相合成器中封闭未反应的氯。用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30分钟，用DMF洗脱6次。将合成的L1(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，在室温下反应过夜，并用DMF洗脱6次，得到Lys-L1。
用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30分钟，再次将合成的L1(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，在室温下反应过夜，并用DMF洗脱6次，得到Lys-(L1) 2。
再次用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30分钟，再次将合成的L1(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，在室温下反应过夜，并用DMF洗脱6次，得到Lys-(L1) 3。
再次用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30分钟，再次将合成的L1(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，在室温下反应过夜，并用DMF洗脱6次，得到Lys-(L1) 4。
再次用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30分钟，再次将合成的L1(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，在室温下反应过夜，并用DMF洗脱6次，得到Lys-(L1) 5。
分别将Lys-L1、Lys-(L1) 2、Lys-(L1) 3、Lys-(L1) 4、Lys-(L1) 5用二甲基亚砜(DMSO)洗脱3次。向FA(2.2g，5.0mmol)的DMSO(40ml)溶液中加入溶于DMSO(10ml)中的1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(479.3mg，2.5mmol)和N-羟基丁二酰亚胺(NHS)(575.5mg,5.0mmol)并避光反应1h，得到化合物FA-NHS。将含有FA-NHS的反应液直接加入到分别偶联了Lys-L1、Lys-(L1) 2、Lys-(L1) 3、Lys-(L1) 4和Lys-(L1) 5的固相合成器中，然后分别加入DIEA(680.0μL,4.0mmol)并室温避光反应过夜。用DMSO洗6次，再用DCM洗脱6次，然后加入8mL洗脱液(三氟乙酸(TFA)/三异丙基硅烷(Tris)/水＝95:2.5:2.5)并室温下洗脱1h。吹走洗脱液中的大部分TFA后加无水乙醚进行沉淀，并用无水乙醚洗涤沉淀三次，室温干燥，得到产物Lys-L1-FA、Lys-(L1) 2-FA、Lys-(L1) 3-FA、Lys-(L1) 4-FA和Lys-(L1) 5-FA。每步反应的产物都经高效液相色谱仪(HPLC)检测纯度。
B.合成Lys-(L1) m-Glu-FA(m＝3、5)化合物的通用程序
采用与Lys-(L1) m-FA的合成程序相似的程序合成Lys-(L1) m-Glu-FA(m＝3、5)。如图3所示，在用20％的哌啶(PIP)的DMF溶液脱除Lys-(L1) 3上的Fmoc保护基30分钟后，将Fmoc-Glu-OtBu(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中，并加入到偶联了Lys-(L1) 3的固相合成器中，室温反应过夜，得到Lys-(L1) 3-Glu。用DMF洗脱3次，再用二甲基亚砜(DMSO)洗脱3次。
按照与Lys-(L1) m-FA的合成方法相同的方法，将新鲜制备的含有FA-NHS的反应液直接加入到偶联了Lys-(L1) 3-Glu的固相合成器中，加入DIEA(4.0eq)并室温避光反应过夜。用DMSO洗6次，再用DCM洗脱6次，然后加入8.0mL洗脱液(三氟乙酸(TFA)/三异丙基硅烷(Tris)/水＝95:2.5:2.5)并室温下洗脱1h。吹走洗脱液中的大部分TFA后加无水乙醚进行沉淀，并用无水乙醚洗涤沉淀三次，并室温干燥，得到产物Lys-(L1) 3-Glu-FA。每步反应的产物都经高效液相色谱仪(HPLC)检测纯度。
采用与Lys-(L1) 3-Glu-FA的合成程序相似的程序，在用20％的哌啶(PIP)的DMF溶液脱除Lys-(L1) 5上的Fmoc保护基30分钟后，将Fmoc-Glu-OtBu(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中，并加入到偶联了Lys-(L1) 5的固相合成器中，室温反应过夜，得到Lys-(L1) 5-Glu。用DMF洗脱3次，再用二甲基亚砜(DMSO)洗脱3次。
按照与Lys-(L1) m-FA的合成方法相似的方法，将新鲜制备的含有FA-NHS的反应液直接加入到偶联了Lys-(L1) 5-Glu的固相合成器中，加入DIEA(4.0eq)，并室温避光反应过夜。用DMSO洗6次，再用DCM洗脱6次后，加入8.0mL洗脱液(三氟乙酸(TFA)/三异丙基硅烷(Tris)/水＝95:2.5:2.5)并室温下洗脱1h。吹走洗脱液中的大部分TFA后加无水乙醚进行沉淀，并用无水乙醚洗涤沉淀三次，室温干燥，得到产物Lys-(L1) 5-Glu-FA。每步反应的产物都经高效液相色谱仪(HPLC)检测纯度。
C.Lys-(L2) 3-Glu-FA化合物的合成
采用与Lys-(L1) 3-Glu-FA的合成程序相似的程序。如图4所示，在合成前1h，用二氯甲烷对载量为1.0mmol/g的三苯基氯树脂(5.0mmol)进行溶胀处理。将Boc-Lys(Fmoc)-OH(1.0g,1.0mmol)和DIEA(680.0μL,4.0mmol)溶解于DCM(10.0mL)中并加入到固相合成器中，室温反应过夜。用DCM洗6次，然后将配制的封闭液(DCM：MeOH：DIEA＝17:1:2)加入到固相合成器中封闭未反应的氯。在用20％的哌啶(PIP)的DMF溶液脱除Fmoc保护基30min并用DMF洗脱6次后，将商购的L2(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中并加入到固相合成器中，室温反应过夜，用DMF洗脱6次，得到Lys-L2。重复上述操作两次，得到Lys-(L2) 3。
用20％的哌啶(PIP)的DMF溶液脱除Lys-(L2) 3上的Fmoc保护基30分钟后，将Fmoc-Glu-OtBu(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中，并加入到偶联了Lys-(L2) 3的固相合成器中，室温反应过夜，得到Lys-(L2) 3-Glu。用DMF洗脱3次，再用二甲基亚砜(DMSO)洗脱3次。按照与Lys-(L1) m-FA的合成方法相似的方法，将新鲜制备的含有FA-NHS的反应液直接加入到偶联了Lys-(L2) 3-Glu的固相合成器中，加入DIEA(4.0eq)，并室温避光反应过夜。用DMSO洗6次，再用DCM洗脱6次后，加入8.0mL洗脱液(三氟乙酸(TFA)/三异丙基硅烷(Tris)/水＝95:2.5:2.5)并室温下洗脱1h。吹走洗脱液中的大部分TFA后加无水乙醚进行沉淀，并用无水乙醚洗涤沉淀三次，室温干燥，得到产物Lys-(L2) 3-Glu-FA。每步反应的产物都经高效液相色谱仪 (HPLC)检测纯度。
D.Lys-(L1) 3-Asp-FA化合物的合成
如图5所示，采用与Lys-(L1) 3-Glu-FA的合成程序相似的程序，用20％的哌啶(PIP)的DMF溶液脱除Lys-(L1) 3上的Fmoc保护基30分钟后，将Fmoc-Asp-OtBu(1.5eq)、缩合剂O-苯并三氮唑-四甲基脲六氟磷酸酯(HBTU)(2.0eq)、1-羟基苯并三唑(HOBt)(2.0eq)和DIEA(4.0eq)溶于DMF中，并加入到偶联了Lys-(L1) 3的固相合成器中，室温反应过夜，得到Lys-(L1) 3-Asp。用DMF洗脱3次，再用二甲基亚砜(DMSO)洗脱3次。按照与Lys-(L1) m-FA的合成方法相似的方法，将新鲜制备的含有FA-NHS的反应液直接加入到偶联了Lys-(L1) 3-Asp的固相合成器中，加入DIEA(4.0eq)，并室温避光反应过夜。用DMSO洗6次，再用DCM洗脱6次，然后加入8.0mL洗脱液(三氟乙酸(TFA)/三异丙基硅烷(Tris)/水＝95:2.5:2.5)，并室温下洗脱1h。吹走洗脱液中的大部分TFA后加无水乙醚进行沉淀，并用无水乙醚洗涤沉淀三次，室温干燥，得到产物Lys-(L1) 3-Asp-FA。每步反应的产物都经高效液相色谱仪(HPLC)检测纯度。
E.Lys-(L1) m-FA(m＝1,2,3,4,5)、Lys-(L1) 3-Glu-FA、Lys-(L1) 5-Glu-FA、Lys-(L2) 3-Glu-FA和Lys-(L1) 3-Asp-FA化合物与Pyro的偶联
Pyro-NHS的合成：Pyro(534.3mg,1.0mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐(EDC)(1.9g,10.0mmol)和N-羟基丁二酰亚胺(NHS)(1.2g,10.0mmol)加入到DMF(60.0mL)中，并室温避光反应过夜。加入水，用DCM稀释并萃取，将有机相用水洗3次，饱和食盐水洗，无水硫酸镁(MgSO 4)干燥，旋蒸除去DCM，得到黑色固体(788.0mg)，产物未经纯化直接用于下一步反应。
分别将Lys-L1-FA、Lys-(L1) 2-FA、Lys-(L1) 3-FA、Lys-(L1) 4-FA、Lys-(L1) 5-FA、Lys-(L1) 3-Glu-FA、Lys-(L1) 5-Glu-FA、Lys-(L2) 3-Glu-FA或Lys-(L1) 3-Asp-FA(1.0eq)溶于DMSO中，再加入DIEA(5.0eq)和Pyro-NHS(0.9eq)，并室温避光反应过夜。向反应液中加入无水乙醚进行沉淀，用无水乙醚洗涤沉淀三次，再用DCM洗涤至没有Pyro-NHS为止，得到产物。产物经HPLC检测纯度，并用HRMS表征最终产物的分子量。
Pyro-Lys-L1-FA：高分辨质谱(MALDI-TOF)荷质比C 72H 91N 15O 15 2+for[M/2+H] +，理论分子量702.8332，我们发现702.8391；
Pyro-Lys-(L1) 2-FA：高分辨质谱(MALDI-TOF)荷质比C 86H 117N 17O 21 2+for[M/2+H] +，理论分子量861.9227，我们发现861.9300；
Pyro-Lys-(L1) 3-FA：高分辨质谱(MALDI-TOF)荷质比C 100H 143N 19O 27 2+for[M/2+H] +，理论分子量1021.0122，我们发现1021.0115；
Pyro-Lys-(L1) 4-FA：高分辨质谱(MALDI-TOF)荷质比C 114H 170N 21O 33 2+for[M/2+H] +，理论分子量1180.1018，我们发现1180.1071；
Pyro-Lys-(L1) 5-FA：高分辨质谱(MALDI-TOF)荷质比C 128H 195N 23O 39 3+for[M/3+H] +，理论分子量893.1275，我们发现893.1352；
Pyro-Lys-(L1) 3-Glu-FA：高分辨质谱(MALDI-TOF)荷质比C 105H 159N 20O 30 2+for[M/2+H] +，理论分子量1085.5335，我们发现1085.5388；
Pyro-Lys-(L1) 5-Glu-FA：高分辨质谱(MALDI-TOF)荷质比C 133H 203N 24O 42 3+for[M/3+H] +，理论分子量936.1417，我们发现936.1480；
Pyro-Lys-(L2) 3-Glu-FA：高分辨质谱(MALDI-TOF)荷质比C 108H 157N 17O 33 2+for[M/2+H] +，理论分子量1111.0565，我们发现1111.0617；
Pyro-Lys-(L1) 3-Asp-FA：高分辨质谱(MALDI-TOF)荷质比C 104H 146N 20O 30 2+for [M/2+H] +，理论分子量1078.5257，我们发现1078.5294。
实施例3.Pyro-Lys-(L1) m-FA(m＝1,2,3,4,5)、Pyro-(L1) 3-Glu-FA和Pyro-(L1) 5-Glu-FA的光谱性质(图7A和图7B，表1)
荧光激发和发射光谱：
荧光发射光谱的记录扫描范围从波长600nm到800nm，激发波长为680nm。荧光激发光谱的记录扫描范围从波长450nm到670nm，发射波长为668nm。将样品均配制成浓度为2.0μM的DMSO溶液。激发和发射的狭缝宽度分别为1nm和2nm。图7A是Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA在DMSO溶液中的荧光激发和发射光谱。由图7A和表1可知，连接部分和FA部分对Pyro部分的光学性能没有明显影响，所有化合物分别在约668nm和672nm处显示最大荧光激发带和发射带。
表1Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA的光谱数据
Figure PCTCN2020094308-appb-000024
UV-Vis光谱：
利用美国Cary 5000型紫外分光光度计对Pyro和Pyro-Lys-(L1) 3-Glu-FA进行紫外吸收光谱的测定。将样品均配制成浓度为10μM的溶液，溶剂分别为DMSO、含有0.05％Tween-80的磷酸缓冲液(PBS)、含有1％聚氧乙烯蓖麻油(CEL)的磷酸缓冲液(PBS)和PBS溶液(其中各种不同溶剂中DMSO的含量均为0.1％)。在室温下，扫描波长范围从300nm到800nm，分辨率为1nm，扫描速率为每分钟600nm。
通过紫外吸收光谱对Pyro-Lys-(L1) 3-Glu-FA和Pyro进行表征。在DMSO中二者均有较强的吸收峰，吸收波长为λ＝680nm，表现出了典型的非聚集形式，严格符合Beer–Lambert定律。与Pyro相比，Pyro-Lys-(L1) 3-Glu-FA表现出很好的水溶性。如图8A所示，Pyro在PBS溶液中发生了聚集，吸收峰变宽。然而，Pyro在含有0.05％Tween-80的PBS溶液中和在含有1％聚氧乙烯蓖麻油(CEL)的PBS溶液中的光谱图与其在DMSO中的光谱图相似，其吸收光谱显示几乎没有聚集。Pyro-Lys-(L1) 3-Glu-FA在含有0.05％Tween-80的PBS溶液、含有1％聚氧乙烯蓖麻油(CEL)的PBS溶液和PBS溶液中的光谱图都与其在DMSO中的光谱图类似。
以上结果表明，引入连接部分和羧基官能团均能够显著提高Pyro的水溶性，并帮助解决了Pyro在水溶液中聚集的问题。由于只有消除聚集的光敏剂才有光活性，因此，消除光敏剂聚集在生物应用中非常重要。
单线态氧量子产率测定：
使用Pyro作为参比化合物，使用单线态氧淬灭剂1,3-二苯基异苯并呋喃(DPBF，25μM)测量光敏剂在DMSO中的单线态氧量子产率。DPBF能够与单线态氧不可逆地 结合，从而造成DPBF在417nm处的吸光度值下降。通过测量DPBF在417nm处的吸光度值变化，间接检测体系内单线态氧的产生。将测试化合物Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA均配制成10μM的DMSO溶液，并取2mL溶液加入到石英比色皿中，再将DPBF加入到测试样品中并混匀。使用波长为680nm的LED灯照射，分别在光照0s、10s、20s、30s、40s、50s、6s和70s后用紫外分光光度计进行扫描，扫描波长范围从350nm到800nm，分辨率为1nm，扫描速率为每分钟600nm。通过将DPBF在417nm处的降低的吸光度值对照射时间进行线性拟合，如图7B所示。根据以下公式计算单线态氧量子产率：
Figure PCTCN2020094308-appb-000025
其中，ΦStdΔ是参比化合物Pyro在DMSO中的单线态氧量子产率，ΦStdΔ＝0.52、Rs.m.DPBF和RStd DPBF分别是测试化合物和参比化合物在417nm处的吸光度值随照射时间降低的线性斜率，AStd 680和As.m.680分别是测试化合物和参比化合物在680nm处的吸光度值。如表1所示，所有的测试化合物在DMSO中都具有较高的单线态氧量子产率，尤其是化合物Pyro-Lys-(L1) 3-Glu-FA产生单线态氧的能力最强。
将在水溶液中的单线态氧量子产率数据(DMSO含量为0.1％)作为参考。在水溶液中的单线态氧测定用ABDA(9，10-蒽基-双(亚甲基)二丙二酸，50nM)作为指示剂。如图8C和8D所示，在不同的光照持续时间下记录400nm处的衰减速率，根据以下公式得到在水溶液中的单线态氧量子产率：
Figure PCTCN2020094308-appb-000026
其中，K RB和K PS分别是参比化合物(RB)和测试化合物在含1％CEL的PBS溶液、含0.05％Tween-80的PBS溶液和PBS溶液中的分解速率常数，其中RB指的是Rose Bengal，其在水溶液中测得的紫外吸收数据作为对照组数据)。A RB和A PS分别代表由参比化合物(RB)和测试化合物吸收的光，其通过对在400-700nm的波长范围内的吸收带进行积分来确定。Ф RB是RB的单线态氧量子产率，在水溶液中为0.75。如图8C所示，化合物Pyro在含1％CEL的PBS溶液和含0.05％Tween-80的PBS溶液中都具有较高的单线态氧量子产率，但是由于Pyro较差的水溶性，其在纯PBS溶液中的单线态氧量子产率很低；而化合物Pyro-Lys-(L1) 3-Glu-FA在所有溶液中都具有较高的单线态氧量子产率(见图8D所示)。
单线态氧量子产率是光动力治疗中影响细胞杀伤力的最主要因素，因此提高生理溶液中的单线态氧量子产率对光动力治疗非常有帮助。
实施例4.不同光敏化合物在细胞水平上的药物活性的评价
用DMSO将Pyro、Pyro-Lys-(L1) m-FA(m＝1、2、3、4、5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA、Pyro-Lys-(L1) 3-Asp-FA和替莫泊芬均配制成10mM母液。在细胞水平上评价药物活性时，各化合物的稀释液中的DMSO含量均低于千分之一。
细胞的复苏和培养
从液氮中取出KB细胞(人口腔表皮癌细胞系)、A431细胞(人皮肤癌细胞系)、NCI-N87细胞(人胃癌细胞系)、A549细胞(人肺癌细胞系)和LO2细胞(人正常肝细胞系)，并置于37℃的水浴锅中迅速解冻，然后以1000rpm/min离心5min，弃去上清液， 加入预热的无叶酸的完全培养基(向无叶酸的基础培养基(含L-谷氨酸和酚红、无叶酸)中添加10％的血清和1％的抗生素，下文简称“培养基”)，并置于5％CO 2、37℃培养箱中培养过夜。第二天更换培养基。继续培养至细胞铺满皿底。传代培养2-3次。
1.Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA在KB细胞水平上药物活性的评价
将KB细胞以1×10 4细胞/孔的密度铺于96孔板中，并放置于37℃、5％的二氧化碳培养箱中培养过夜。将上层清液去除后，向每孔中加入用新鲜的培养基配制的含不同药物浓度的Pyro-Lys-(L1) m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA或Pyro-Lys-(L1) 3-Asp-FA，并放回37℃、5％二氧化碳培养箱中，使药物与细胞孵育4h。将细胞培养板取出，用660nm光源在距离培养板约2cm处给予40mW/cm 2的光辐射剂量10min。然后，将96孔细胞培养板放回到细胞培养箱中继续培养过夜。用新鲜的培养基配制0.5mg/ml的MTT溶液。将96孔板中的培养基去除后，每孔中加入100μL的含有MTT的培养基，再放回细胞培养箱中继续培养4h，然后去除细胞培养液，每孔中加入100μL的DMSO，室温摇晃10min，在多功能酶联免疫仪中测定490nm的吸光度。根据吸光度计算细胞的存活率，然后绘制细胞存活率和药物浓度之间关系的药物活力抑制曲线，并计算相应的半数抑制浓度(IC 50值)。每个实验组在5个孔上实施，数据表示为平均值±SEM。
实验结果与结论：分别合成了具有不同链长且任选地具有Glu片段的连接部分的化合物Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)或Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA。即，Pyro-Lys-L1-FA、Pyro-Lys-(L1) 2-FA、Pyro-Lys-(L1) 3-FA、Pyro-Lys-(L1) 4-FA、Pyro-Lys-(L1) 5-FA、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA，分子量分别是1403.6663、1721.8453、2040.0244、2358.2035、2676.3826、2169.0670和2805.4252。通过细胞毒性实验(MTT测定)来研究Pyro-Lys-(L1)m-FA(m＝1、2、3、4、5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA的药物活性变化。
在第201710550348.1号中国专利申请中，通过向将小分子靶向配基叶酸与光敏剂Pyro连接的含有半胱氨酸的连接链中引入高度亲水的不同链长的聚乙二醇，改善了Pyro-FA的性能。合成后的化合物具有良好的水溶性，并增强了对肿瘤的靶向富集能力，改善了光敏剂在体内的代谢分布特性，增强了动物水平上的光动力治疗的效果。使用细胞膜表面叶酸受体表达量高的KB(人口腔表皮癌细胞)进行研究。如图9所示，发现L1链的引入显著提高药物活性。在第201610628964.X号中国专利中，同样在KB细胞水平上的药物活性研究中，Pyro-FA的IC 50值约为500nM；在第201710550348.1号中国专利申请中，筛选出的最优选的化合物Pyro-PEG1K-FA对阳性KB细胞的IC 50值约为136nM。如表2所示，当引入L1后，Pyro-Lys-L1-FA的IC 50值为53.32nM，Pyro-Lys-(L1) 2-FA的IC 50值为23.15nM。随着继续增加L1，即，测试化合物分别是Pyro-Lys-(L1) 3-FA、Pyro-Lys-(L1) 4-FA、Pyro-Lys-(L1) 5-FA时，药物活性变化不再明显。在引入Glu(谷氨酸)基团后，Pyro-Lys-(L1) 3-Glu-FA的IC 50值为1nM，Pyro-Lys-(L1) 5-Glu-FA的IC 50值为约22nM。与第201710550348.1号中国专利中的最优选化合物Pyro-PEG1K-FA相比，本申请的Pyro-Lys-(L1) 3-Glu-FA对阳性KB细胞的半数抑制浓度(IC 50值)降低了上百倍，即对阳性细胞系的杀伤力提高了上百倍。
在Pyro-Lys-(L1) 3-Glu-FA的基础上，将L1替换成L2，得到化合物Pyro-Lys-(L2) 3-Glu-FA(分子量为2220.1130)，其药物活性与Pyro-Lys-(L1) 3-Glu-FA相似，IC50值约为7.6nM。将Glu片段替换成Asp片段(天冬氨酸片段)，得到化合物Pyro-Lys-(L1) 3-Asp-FA(分子量为2155.0514)，IC50值约为12.03nM”。
表2 Pyro-Lys-(L1)m-FA(m＝1、2、3、4、5)、Pyro-Lys-(L1) 3-Glu-FA、Pyro-Lys-(L1) 5-Glu-FA、Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA对KB细胞的药物活性
Figure PCTCN2020094308-appb-000027
2.Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别在KB和A549细胞系中的选择性内吞实验
分别取对数期的叶酸受体高表达的KB细胞和叶酸受体低表达的A549细胞。在超净工作台中，用0.05％-胰酶-EDTA(Gibco)消化KB和A549细胞。在显微镜下计数后，配制成一定浓度的细胞悬浮液，然后在6孔板中以每孔5×10 4个细胞的密度加入细胞悬浮液，并在二氧化碳培养箱内培养16h。将Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬与Pyro(作为对照组品)分别配制成10mM的DMSO母液，再用相应的完全培养基稀释成不同药物浓度(0.1nM、1nM、10nM、20nM和100nM)的稀释液，在细胞培养箱中继续避光培养4h。除去6孔板中含药物的培养基，并用PBS洗1遍，然后每孔中加入200μL胰酶在37℃下消化3～5min，并在4℃下以1000rpm/min离心3min。用200μL PBS重选细胞，并用BD FACSCalibur流式细胞仪测定荧光强度(λex＝635nm；λem＝645-677nm)。
实验结果与结论：利用光敏剂具有荧光激发和发射的性质直接检测光敏剂被细胞吸收的含量。根据上文的Pyro-Lys-(L1) 3-Glu-FA的激发和发射光谱调节流式细胞仪的参数，通过流式细胞仪直接检测Pyro-Lys-(L1) 3-Glu-FA在叶酸受体高表达的KB细胞系与叶酸受体低表达的A549细胞系中被选择性吸收的对比。如图10所示，在低浓度的药物剂量(0.1nM)下，Pyro-Lys-(L1) 3-Glu-FA能够被KB细胞吸收，并且随着药物浓度的增加，被KB细胞内吞的量逐渐增加，而在高达100nM的浓度下，Pyro-Lys-(L1) 3-Glu-FA被A549细胞吸收的量非常少，在两种细胞系中所吸收的Pyro-Lys-(L1) 3-Glu-FA的量有着显著的差异。作为对照组的Pyro和替莫泊芬，在叶酸受体高表达KB细胞系和叶酸受体低表达A549细胞系中都没有体现出明显的被选择性吸收的差异。
3.Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别与叶酸对KB细胞系活性的竞争性抑制实验
按照与上文所述方法相似的方法，将KB细胞以5×10 4细胞/孔的密度铺于6孔板中，并在二氧化碳培养箱内培养16h。配制含1mM叶酸的培养基，并加入到6孔板的孔中，与细胞孵育4h，然后更换分别含有不同浓度(10nM、100nM、250nM和500nM)的Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬的新鲜培养基，并在细胞培养箱中继续孵育4h，随后置于660nm的光源下，照射10min。在细胞培养箱中继续避光培养12h后，用胰酶消化细胞，并在流式管中在4℃下以1000rpm/min离心3min。对于 每个流式管，用100μL的1×结合缓冲液重悬细胞，并分别加入1μL PI工作液和4μL YF488-Annexin V，在冰上避光孵育15min。然后，向每管中再次加入400μL的1×结合缓冲液，通过BD FACSCalibur流式细胞仪检测细胞凋亡情况。YF488-Annexin V用488nm激光激发，检测药物的荧光发射光谱在530nm处(FITC通道)，PI通道发射光谱约在617nm处。
实验结果与结论：为了证实叶酸和叶酸受体在介导药物的结合和内吞过程中的作用，即，叶酸配基的靶向结合能力，将过量的小分子叶酸(1mM)提前加入到细胞培养基中，通过小分子叶酸的竞争性抑制作用使细胞对Pyro-Lys-(L1) 3-Glu-FA的摄取减少，从而达到竞争性抑制Pyro-Lys-(L1) 3-Glu-FA的细胞毒性作用。如图11所示，叶酸分子能够明显抑制Pyro-Lys-(L1) 3-Glu-FA对KB细胞的杀伤能力，而Pyro和替莫泊芬对细胞的杀伤能力并没有受到额外加入的叶酸分子的影响。这些实验结果证实，在给予光辐射的条件下，Pyro-Lys-(L1) 3-Glu-FA通过与叶酸受体的结合并经受体介导的内吞作用进入细胞，从而发挥其光动力活性。
4.Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬在不同细胞系水平上的药物活性评价及暗毒性分析
将KB细胞、A431细胞、NCI-N87细胞、A549细胞和LO2细胞分别以1×10 4细胞/孔的密度铺于96孔板中，并在培养箱中培养16h。分别将相应浓度的Pyro、Pyro-Lys-(L1) 3-Glu-FA或替莫泊芬加入培养孔中，并将培养板置于细胞培养箱中培养4h，再置于660nm的光源下，以40mW/cm 2的光强照射10min，然后在细胞培养箱中继续过夜培养。用含MTT的新鲜培养基替换原来的培养基，与细胞共同孵育4h，然后对每孔用100μL的二甲基亚砜置换培养基，测定490nm的吸光度，每个实验组在5个孔上实施，数据表示为平均值±SEM。
暗毒性是指在没有光辐射激发光敏剂的情况下，光敏剂会对细胞产生的毒性。作为理想的光敏剂应无明显的无激发暗毒性。我们使用体外细胞活力抑制实验来评估Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬的暗毒性水平。按照上述方法将五种细胞铺于96孔板中并培养16h，然后分别加入相应浓度的Pyro、Pyro-Lys-(L1) 3-Glu-FA或替莫泊芬。将培养板置于细胞培养箱中培养4h，用新鲜培养基更换含药物的培养基，并将培养基在避光条件下放置过夜。通过上述细胞活性实验，使用MTT法测定黑暗条件下药物对细胞活力的影响。
实验结果与结论：具有不同叶酸受体表达量的各个细胞系被用来评价Pyro-Lys-(L1) 3-Glu-FA的药物活性，并在相同的实验条件下，Pyro和替莫泊芬对不同细胞系的杀伤效果作为本实验的对照组进行评价。如图12所示，在使用的药物浓度范围内，Pyro-Lys-(L1) 3-Glu-FA、Pyro和替莫泊芬在给予的特定光照条件下对不同细胞系的杀伤能力体现出剂量依赖性关系。
表3 Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬在不同细胞系上的光毒性和暗毒性
Figure PCTCN2020094308-appb-000028
Figure PCTCN2020094308-appb-000029
Figure PCTCN2020094308-appb-000030
如表3所示，在给予24J/cm 2的光照条件下，Pyro-Lys-(L1) 3-Glu-FA对叶酸受体表达量较高的KB细胞系、A431细胞系和NCI-N87细胞系的杀伤能力明显高于叶酸受体表达量相对较低的A549细胞系和LO2细胞系。
通过软件计算，可知Pyro-Lys-(L1) 3-Glu-FA对KB细胞系的IC 50值为1.403nM，对A431细胞系的IC 50值为103.3nM，对NCI-N87细胞系的IC 50值为94.48nM，而Pyro-Lys-(L1) 3-Glu-FA对A549细胞系的IC 50值为2864nM，对正常的人肝细胞系LO2的IC 50值为2330nM。Pyro-Lys-(L1) 3-Glu-FA对KB细胞系的杀伤活性是对A549细胞系杀伤活性的2000倍和对LO2细胞系杀伤活性的1660倍。而对照组Pyro和替莫泊芬对不同细胞系的IC 50值均在65～80nM和100～200nM之间，仅有微弱的差异，而这种微弱的差异来自于不同细胞系对Pyro和替莫泊芬的敏感性不同。
因此，靶向配基—叶酸的引入使得Pyro-Lys-(L1) 3-Glu-FA能够对具有不同叶酸受体表达量的细胞系产生选择性杀伤能力，这有利于降低光敏药物对正常组织的杀伤作用。
在相同的药物浓度范围内，Pyro-Lys-(L1) 3-Glu-FA在没有光照情况下对不同细胞系均没有体现出明显的细胞毒性，证明Pyro-Lys-(L1) 3-Glu-FA符合理想的光敏剂在无光照情况下没有毒副作用的重要属性。
实施例5.不同化合物在小鼠体内的分布特点及对肿瘤的治疗效果的评价
小鼠皮下肿瘤模型的建立：
小鼠的皮下肿瘤模型分别为KB肿瘤模型、A431肿瘤模型和NCI-N87肿瘤模型。6～8周龄的雌性BALB/c裸鼠购自北京维通利华实验动物技术有限公司，并一直饲养在SPF级的环境中。具体的实验方法如下：首先按照小鼠的数量计算所需的肿瘤细胞的数量，合理地大量培养相应的肿瘤细胞。在本实验的各个肿瘤模型中，每只小鼠需3×10 6个细胞。在所培养的细胞占培养皿底板的约80％的空间后，将细胞用与传代类似的方式进行消化，并在预先冷却至4℃的离心机中以1000rpm离心3分钟。弃去上清液，将细胞用PBS悬浮均匀，然后吸取10μL并置于细胞计数板中进行计数。根据计数结果配制成细胞悬液，按每只小鼠皮下注射100μL的量计算。
小鼠皮下肿瘤接种方法：将小鼠用15％异氟烷麻醉后，在小鼠的背部右侧用75％酒精擦拭消毒，然后用注射器吸取100μL(3×10 6个细胞)肿瘤细胞悬液，并将细胞注入小鼠的皮下，然后将针尖倒转，拔出小鼠体内，并做好标记。在5天后，每2天检测一次小鼠肿瘤的体积大小情况，并记录肿瘤体积和小鼠的体重变化。
1.Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA在KB移植瘤小鼠体内随时间分布的药物代谢过程
由于光敏药物的荧光特性，因此可以利用Xenogen IVIS小动物活体成像系统来研究光敏药物在体内的分布情况。构建小鼠皮下肿瘤KB肿瘤模型(每组5只小鼠)。在小鼠的皮下肿瘤长到约200mm 3时，先将小鼠用15％异氟烷短时麻醉，然后放置于Xenogen IVIS小动物活体成像仪中，用λex＝640nm、λem＝695-770nm、曝光时间为5s的参数扫描各组小鼠。将此时小鼠肿瘤部位和身体其余部位的自发荧光值作为背景扣除。然后，将小鼠分别通过尾静脉注射50nmol的Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA(每只小鼠静脉给药的DMSO含量为2.5％)，并由注射时间点作为起点，用活体成像系统扫描给药后0.5h、1h、2h、3h、4h和6h的药物分布情况。
实验结果与结论：利用小动物活体成像系统研究Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA在KB肿瘤模型小鼠体内的药物分布随时间的变化。当经尾静脉给予50nmol的Pyro-Lys-(L1)m-FA(m＝1、2、3、4或5)、Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA后，在不同时间点(0.5h、1h、2h、3h、4h和6h)分别用15％异氟烷将小鼠麻醉，扫描此时小鼠体内的药物分布。如图13所示，对于Pyro-Lys-L1-FA、Pyro-Lys-(L1) 2-FA、Pyro-Lys-(L1) 3-FA、Pyro-Lys-(L1) 4-FA和Pyro-Lys-(L1) 5-FA而言，随着L1的引入和增加，化合物在肿瘤组织中的分布比例越来越多。当给予小鼠相同剂量的Pyro-Lys-(L1) 3-Glu-FA或Pyro-Lys-(L1) 5-Glu-FA后，化合物可在给药后1h快速集中于小鼠的肝脏、肾脏和肿瘤组织部位，并随着时间的延长，药物被逐渐代谢；在给药后2h，肝脏中的化合物含量明显降低，化合物主要富集于肿瘤部位。
该实验结果表明，在注射相同剂量药物的情况下，Pyro-Lys-(L1) 3-Glu-FA和Pyro-Lys-(L1) 5-Glu-FA均具有较强的快速的在肿瘤部位富集的能力。
2.Pyro-Lys-(L2) 3-Glu-FA和Pyro-Lys-(L1) 3-Asp-FA在KB移植瘤小鼠体内随时间分布的药物代谢过程
利用上述方法构建小鼠皮下肿瘤KB肿瘤模型(每组5只小鼠)。在小鼠的皮下肿瘤长到约200mm 3时，给予小鼠相同剂量(50nmol)的Pyro-Lys-(L2) 3-Glu-FA或Pyro-Lys-(L1) 3-Asp-FA(每只小鼠静脉给药的DMSO含量小于2.5％)，并以注射时间点作为起点，用活体成像系统扫描尾静脉给药后1h、2h、4h、6h、9h和24h时药物在小鼠体内的分布情况。
实验结果与结论：如图14所示，Pyro-Lys-(L2) 3-Glu-FA在给药后2h时可快速富集于肿瘤和肝脏组织，在给药后4h～6h时肝脏中的含量明显降低，化合物主要富集于肿瘤组织中，在给药24h后，小鼠体内的药物几乎全都通过代谢排出。Pyro-Lys-(L1) 3-Asp-FA在给药后2h时也可快速富集于肿瘤组织部位，其在小鼠体内的代谢速度比Pyro-Lys-(L2) 3-Glu-FA快。
3.Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬分别在KB、A431或NCI-N87移植瘤小鼠体内随时间分布的药物代谢过程以及它们在各器官、肿瘤和肌肉组织中的富集(Ht:心；Lv:肝；Sp:脾；Lu:肺；Kn:肾；Tm:肿瘤；Ms:肌肉)
用多种荷瘤的雌性BALB/c裸鼠研究Pyro-Lys-(L1) 3-Glu-FA的药物代谢特点，并用Pyro和替莫泊芬作为对照组。
在小鼠后背部接种KB细胞或A431细胞或NCI-N87细胞(每组5只小鼠)。在肿瘤体积长至约200mm 3时，按照上述方法扫描未作处理的小鼠以作为背景对照。然后通过尾静脉分别向各组小鼠注射50nmol的Pyro、Pyro-Lys-(L1) 3-Glu-FA或替莫泊芬(每 只小鼠静脉给药的DMSO含量小于2.5％)，并以注射时间点作为起点。对Pyro和Pyro-Lys-(L1) 3-Glu-FA组小鼠，分别扫描给药后0.5h、1h、2h、3h、4h和6h小鼠体内的药物分布情况，而对于替莫泊芬组小鼠，扫描给药后0.5h、1h、2h、3h、4h、6h、12h、1d、2d、3d、4d、6d和8d小鼠体内的药物分布情况。
给药后24h时，处死小鼠，将各组小鼠的各器官组织、肿瘤组织以及肌肉组织取出，并通过活体成像仪扫描各组小鼠的各器官组织、肿瘤组织及肌肉组织中所含有的化合物量。利用活体成像系统的软件，计算肿瘤部位的平均荧光强度和各个组织的总平均荧光强度，并计算两者的比值，此比值能够反应光敏药物在肿瘤内的平均富集程度以及该光敏药物在降低皮肤毒性方面的应用潜力。ROI(region of interest)的单位表示为：[p/sec/cm 2/sr]/[μW/cm 2]。
实验结果与结论：使用叶酸受体高表达的KB肿瘤模型和叶酸受体中等表达的A431和NCI-N87肿瘤模型研究Pyro-Lys-(L1) 3-Glu-FA在小鼠体内的分布特点，并以Pyro和替莫泊芬作为对照。如图15A、15B和15C所示，在KB肿瘤模型的荷瘤小鼠中，Pyro-Lys-(L1) 3-Glu-FA在尾静脉注射后1h开始在肿瘤组织中富集，在2h时在小鼠身体的其余部位的荧光背景明显降低，在肿瘤组织中的荧光强度明显高于在周围的皮肤组织和肝脏组织中的荧光强度。
Pyro和替莫泊芬在此肿瘤模型中的分布情况和Pyro-Lys-(L1) 3-Glu-FA截然不同。在尾静脉注射后，Pyro在小鼠的肝脏组织中有明显的大量富集，并随着给药后时间的延长，Pyro在肿瘤组织中没有明显的积累作用，而持续在小鼠的肝脏组织中有大量积累。替莫泊芬在给药前期分布在小鼠体内的荧光并不明显，在给药后2h，在小鼠的肝脏组织中可见荧光，并随着给药后时间的延长，小鼠肝脏组织中的药物含量逐渐增多，并向肿瘤组织转移；在给药后24h时，小鼠肿瘤组织中的药物量达到最大值，并有一部分药物开始向肺组织转移，药物在体内的代谢时间长达8d。
Pyro、Pyro-Lys-(L1) 3-Glu-FA和替莫泊芬在A431及NCI-N87肿瘤模型小鼠中的分布特点与KB肿瘤模型小鼠中的分布特点相似。Pyro-Lys-(L1) 3-Glu-FA在给药后2h时可观察到在肿瘤组织部位有明显的富集，而Pyro一直大量聚集于肝脏组织并在肿瘤组织中未有明显的积累作用，以及替莫泊芬在给药后12h～24h时在肿瘤组织中的积累量达到最大值，并在体内的代谢时间依然长达8d。
另外，各个肿瘤模型的小鼠在给药后24h时被处死，并取出各组小鼠的心、肝、脾、肺、肾及肿瘤和肌肉组织，并直接在动物活体成像仪中进行对比观察。如图15E所示，给药后24h，Pyro-Lys-(L1) 3-Glu-FA在KB、A431和NCI-N87肿瘤组织和肾脏组织中均有大量富集，而在肝脏组织中含量较少。绝大部分的Pyro累积在肝脏组织中。替莫泊芬虽然也聚集在肿瘤组织部位，但也大量地累积在肝、肺、肾等组织中。如图15D所示，在KB、A431和NCI-N87肿瘤小鼠模型中，尾静脉给药后2h时，Pyro-Lys-(L1) 3-Glu-FA在肿瘤组织中的积累量与在解剖出的所有组织中的总积累量的比值均是最高的。
以上实验结果表明，注射相同剂量药物的情况下，Pyro-Lys-(L1) 3-Glu-FA在多种肿瘤模型中均具有较强的肿瘤富集能力，并且在体内的循环时间相对较短。
4.Pyro-Lys-(L1) 3-Glu-FA分别对KB、A431和NCI-N87移植瘤小鼠的肿瘤光动力治疗效果
使用具有不同叶酸受体表达量的KB、A431、NCI-N87肿瘤模型研究Pyro-Lys-(L1) 3-Glu-FA的治疗效果。
将3×10 6个KB细胞皮下接种于小鼠的背部右侧。在大约18d后，小鼠的肿瘤体积达到约200mm 3。将KB移植瘤小鼠随机分为如下5组：(1)200μL PBS/光+(光照)(对 照组)；(2)60nmol Pyro/光-(不光照)；(3)60nmol Pyro/光+(光照)；(4)60nmol Pyro-Lys-(L1) 3-Glu-FA/光-(不光照)；和(5)60nmol Pyro-Lys-(L1) 3-Glu-FA/光+(光照)，每组5只小鼠(每只小鼠静脉给药的DMSO含量为3％)。各光照组给予的光照能量为120J/cm 2(200mW/cm 2，10min)。将尾静脉给药后2h作为小鼠光动力治疗的照射时间点。每两天用游标卡尺记录小鼠肿瘤的体积变化，并记录小鼠的体重变化。当小鼠的肿瘤体积达到1500mm 3时，小鼠被认为死亡并停止记录，持续观察小鼠至95天。
用上述方法分别在小鼠皮下接种3×10 6个A431或NCI-N87肿瘤细胞，并将小鼠随机分为如下三组(每组5只)：(1)PBS/光+(光照)；(2)60nmol Pyro/光+(光照)；和(3)60nmol Pyro-Lys-(L1) 3-Glu-FA/光+(光照)。在尾静脉给药后2h时将660nm波长的光以200mW/cm 2照射肿瘤组织部位10min，并每两天监测小鼠的肿瘤体积和小鼠体重变化。小鼠的肿瘤体积计算公式为：肿瘤体积＝长×宽 2×0.5。
实验结果与结论：如图16A所示，给予光照后，Pyro-Lys-(L1) 3-Glu-FA/光+组的小鼠肿瘤附近出现炎性水肿，2d后水肿情况得到缓解，Pyro-Lys-(L1) 3-Glu-FA/光+组的小鼠肿瘤部位结痂且肿瘤体积开始缩小，两周后痂掉落完全且肿瘤消失，持续观察95天未见复发。而Pyro/光+组的小鼠肿瘤部位炎症较轻，2d后水肿几乎消退，没有结痂现象，肿瘤体积在短时间内得到抑制；但在10d后肿瘤体积快速增长。PBS/光+组的小鼠在光照后皮肤未出现水肿现象，并且肿瘤体积与只给药不光照组(Pyro/光-组和Pyro-Lys-(L1) 3-Glu-FA/光-组)的小鼠的肿瘤体积一样快速增长，在光照或给药后约18d时肿瘤体积很快达到1500mm 3。在没有光照或光敏剂的实验组中，均没有产生对肿瘤体积增长的抑制作用。
用上述方法分别对A431和NCI-N87移植瘤小鼠进行光动力治疗。
Pyro-Lys-(L1) 3-Glu-FA/光+组的小鼠在光照后也出现水肿现象，并且小鼠的肿瘤体积逐渐缩小，但是在第一次光动力治疗后的15～25天内，一些小鼠的肿瘤再次增长。当复发瘤长至约100mm 3时，分别对复发瘤进行第二次给药治疗(同样是尾静脉给药60nmol，给药后2h给予光照能量200J/cm 2)。在之后监测的两个月里，肿瘤没有再出现复发的迹象。
对于Pyro组，用相同的条件进行光动力治疗。如图16B和16C所示，在给予光照后，A431和NCI-N87肿瘤的生长也在短时间内被抑制，但很快就继续增长至1500mm 3。实验结果表明，Pyro-Lys-(L1) 3-Glu-FA具有在肿瘤组织中特异性富集的能力，因此以Pyro-Lys-(L1) 3-Glu-FA为光敏剂的光动力治疗效果优于Pyro的治疗效果。
通过监测小鼠的体重变化来间接监测在光动力治疗过程中测试化合物对小鼠的生理状态造成的影响。如图16A、16B和16C中的第一列图所示，在KB、A431和NCI-N87肿瘤模型小鼠中，Pyro-Lys-(L1) 3-Glu-FA/光+组的小鼠体重均在第一次治疗后10d有降低的趋势，但随后体重慢慢增加，直至跟其余各组的小鼠体重一样，趋于平稳，并呈缓慢上升的趋势。该组小鼠经过一次或两次光动力治疗后存活率均可达到100％。
因此，Pyro-Lys-(L1) 3-Glu-FA是一种高效的具有低毒副作用的可用于抗肿瘤的光动力治疗的光敏剂。
5.Pyro和Pyro-Lys-(L1) 3-Glu-FA对KB移植瘤小鼠的急性毒性试验评价
体重为18～20g的雌性BALB/c裸鼠在给药前禁食6h，然后将小鼠分为三组(每组5只)：(1)5μL DMSO+1μL蓖麻油，避光；(2)500nmol Pyro+1μL蓖麻油，避光；和(3)500nmol Pyro-Lys-(L1) 3-Glu-FA，避光。每隔2d给一次药，共给药4次，每天检测各组小鼠的体重变化，并监测小鼠的生理反应及行为表现。
实验结果与结论：如图17所示，尾静脉注射为500nmol的高剂量药物4次后， 在不给予任何光照的情况下，小鼠的体重呈维持状态，并且没有发生死亡现象。每次给药后，各组小鼠均没有出现恶心、流涎、呕吐等中毒现象，也没有明显的躁动行为。因此，可初步认为光敏剂Pyro和Pyro-Lys-(L1) 3-Glu-FA在小鼠体内的毒性较小，为后续Pyro-Lys-(L1) 3-Glu-FA的开发提供了潜在的可能。
上文对本申请中涉及的一般性描述和对其具体实施方案(包括实施例)的描述不应理解为对本发明技术方案构成限制。在不违背所涉及的发明构成要素的前提下，本领域所属技术人员根据本发明可以对上文一般性描述或/和具体实施方案(包括实施例)中公开的技术特征进行增加、减少或组合，形成属于所属发明的其它技术方案，它们也同样在本发明的保护范围。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (21)

  1. 通式(I)的光敏剂或其药学上可接受的盐：
    A-Lys-(L) m-(B) o-C
    (I)
    其中
    A和C独立地选自叶酸基团(FA)和卟吩型光敏部分(PS)，并且当A和C中的任一个为叶酸基团时，则另一个为卟吩型光敏部分；
    Lys表示
    Figure PCTCN2020094308-appb-100001
    L表示连接子，其独立地选自L1或L2；
    Figure PCTCN2020094308-appb-100002
    B表示酸性氨基酸片段；
    m为1-5的整数；
    o为0或1；以及
    p为4-8的整数。
  2. 如权利要求1所述的光敏剂，其中所述叶酸基团具有如下结构：
    Figure PCTCN2020094308-appb-100003
  3. 如权利要求1或2所述的光敏剂，其中所述卟吩型光敏部分独立地选自Pyro的基团、Ce6的基团、HPPH的基团和Vertoporfin的基团，优选为Pyro的基团，所述Pyro的基团优选地具有结构
    Figure PCTCN2020094308-appb-100004
    Figure PCTCN2020094308-appb-100005
  4. 如权利要求1-3中任一项所述的光敏剂，其中所述酸性氨基酸片段为谷氨酸片段或天冬氨酸片段；所述谷氨酸片段优选地具有结构
    Figure PCTCN2020094308-appb-100006
    以及所述天冬氨酸片段优选地具有结构
    Figure PCTCN2020094308-appb-100007
  5. 如权利要求1-4中任一项所述的光敏剂，m优选为2、3、4或5，更优选为3或5；最优选为3；o优选为1；所述L2中的重复单元-CH 2CH 2O-的个数p为4、5、6、7或8，优选为6。
  6. 如权利要求1所述的光敏剂，其中-(L) m-(B) o-具有如下结构之一：
    Figure PCTCN2020094308-appb-100008
  7. 如权利要求1所述的光敏剂，其中所述通式(I)的光敏剂选自如下化合物之一或其药学上可接受的盐：
    Pyro-Lys-L1-FA：
    Figure PCTCN2020094308-appb-100009
    Pyro-Lys-(L1) 2-FA：
    Figure PCTCN2020094308-appb-100010
    Pyro-Lys-(L1) 3-FA：
    Figure PCTCN2020094308-appb-100011
    Pyro-Lys-(L1) 4-FA：
    Figure PCTCN2020094308-appb-100012
    Pyro-Lys-(L1) 5-FA：
    Figure PCTCN2020094308-appb-100013
    Pyro-Lys-(L1) 5-Glu-FA：
    Figure PCTCN2020094308-appb-100014
    Pyro-Lys-(L1) 3-Glu-FA：
    Figure PCTCN2020094308-appb-100015
    Pyro-Lys-(L2) 3-Glu-FA：
    Figure PCTCN2020094308-appb-100016
    Pyro-Lys-(L1) 3-Asp-FA：
    Figure PCTCN2020094308-appb-100017
  8. 制备权利要求1-7中任一项所述的光敏剂的方法，其包括：
    1)合成连接子L；
    2)使m个连接子L与Lys连接以获得片段Lys-(L) m；
    3)任选地使片段Lys-(L) m与酸性氨基酸片段B连接以获得片段Lys-(L) m-(B) o；
    4)将片段Lys-(L) m-(B) o与叶酸FA连接以获得片段Lys-(L) m-(B) o-FA；以及
    5)将片段Lys-(L) m-(B) o-FA与卟吩型光敏化合物PS连接以获得通式(I)的光敏剂；或者
    6)将片段Lys-(L) m-(B) o与卟吩型光敏化合物连接以获得片段Lys-(L) m-(B) o-PS；以及
    7)将片段Lys-(L) m-(B) o-PS与叶酸FA连接以获得通式(I)的光敏剂；
    其中Lys、L、m、B、o、FA和PS如权利要求1-7中任一项所定义。
  9. 药物组合物，其包含权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐以及药学上可接受的赋形剂。
  10. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在制备用于治疗肿瘤的药物中的用途。
  11. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在制备肿瘤成像诊断试剂中的用途。
  12. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在制备通过光动力疗法来治疗肿瘤的药物中的用途。
  13. 治疗肿瘤的方法，其包括将权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物向有需要的个体给药。
  14. 使肿瘤成像以进行诊断的方法，其包括将权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物向有需要的个体给药。
  15. 通过光动力疗法来治疗肿瘤的方法，其包括将权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物向有需要的个体给药。
  16. 用于治疗肿瘤的权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物。
  17. 用于使肿瘤成像以进行诊断的权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物。
  18. 通过光动力疗法来治疗肿瘤的权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物。
  19. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在治疗肿瘤中的用途。
  20. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在使肿瘤成像以进行诊断中的用途。
  21. 权利要求1-7中任一项所述的光敏剂或其药学上可接受的盐或权利要求9所述的药物组合物在通过光动力疗法来治疗肿瘤中的用途。
PCT/CN2020/094308 2019-06-04 2020-06-04 不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用 WO2020244570A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910481489.1A CN112028898B (zh) 2019-06-04 2019-06-04 不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用
CN201910481489.1 2019-06-04

Publications (1)

Publication Number Publication Date
WO2020244570A1 true WO2020244570A1 (zh) 2020-12-10

Family

ID=73576570

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/094308 WO2020244570A1 (zh) 2019-06-04 2020-06-04 不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用

Country Status (2)

Country Link
CN (1) CN112028898B (zh)
WO (1) WO2020244570A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102895670A (zh) * 2012-11-09 2013-01-30 中国人民解放军第三军医大学野战外科研究所 水溶性分子靶向卟吩光敏剂及其制备方法
CN105343878A (zh) * 2015-11-30 2016-02-24 中国人民解放军第三军医大学第三附属医院 还原敏感型水溶性分子靶向光敏剂及其制备方法和应用
WO2017044443A1 (en) * 2015-09-09 2017-03-16 On Target Laboratories, LLC Synthesis and composition of photodynamic therapeutic agents for the targeted treatment of cancer
CN107226839A (zh) * 2017-07-07 2017-10-03 康宏耀源（天津）科技有限公司 一种rgd多肽偶联的酞菁硅光敏剂的合成和应用
CN107337685A (zh) * 2017-07-07 2017-11-10 康宏耀源（天津）科技有限公司 叶酸靶向Pyro光敏的合成和应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5002962A (en) * 1988-07-20 1991-03-26 Health Research, Inc. Photosensitizing agents
EP2431366B1 (en) * 2009-04-29 2014-04-09 Diatech Korea Co., Ltd. New chlorin e6-folic acid conjugated compound, preparation method thereof, and pharmaceutical composition containing the same for treatment of cancer
CN105749280A (zh) * 2016-04-07 2016-07-13 沈阳大学 一种协同化疗与光动力治疗的肿瘤靶向纳米给药系统的制备及应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102895670A (zh) * 2012-11-09 2013-01-30 中国人民解放军第三军医大学野战外科研究所 水溶性分子靶向卟吩光敏剂及其制备方法
WO2017044443A1 (en) * 2015-09-09 2017-03-16 On Target Laboratories, LLC Synthesis and composition of photodynamic therapeutic agents for the targeted treatment of cancer
CN105343878A (zh) * 2015-11-30 2016-02-24 中国人民解放军第三军医大学第三附属医院 还原敏感型水溶性分子靶向光敏剂及其制备方法和应用
CN107226839A (zh) * 2017-07-07 2017-10-03 康宏耀源（天津）科技有限公司 一种rgd多肽偶联的酞菁硅光敏剂的合成和应用
CN107337685A (zh) * 2017-07-07 2017-11-10 康宏耀源（天津）科技有限公司 叶酸靶向Pyro光敏的合成和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SIBRIAN-VAZQUEZ ETC.: "Synthesis and Properties of Cell-Targeted Zn(II)-Phthalocyanine-Peptide conjugates", BIOCONJUGATE CHEMISTRY, vol. 18, no. 2,, 2 June 2007 (2007-06-02), pages 410 - 420, XP002633589 *

Also Published As

Publication number Publication date
CN112028898B (zh) 2023-06-23
CN112028898A (zh) 2020-12-04

Similar Documents

Publication Publication Date Title
EP0200218B1 (en) Tetrapyrrole therapeutic agents
EP1420824B1 (en) Sulfonated chlorines as photosensitizer
EP0213272A2 (en) New tetrapyrrole polyaminomonocarboxylic acid therapeutic agents
JPS61129163A (ja) 新規なテトラピロ−ル化合物
EP3201201B1 (en) Specifically meso-substituted porphyrins and chlorins for photodynamic therapy
KR102245556B1 (ko) 신규 클로린 e6 유도체 및 이의 약학적으로 허용가능한 염, 이의 제조방법 및 응용
Bakleh et al. An efficient route to VEGF-like peptide porphyrin conjugates via microwave-assisted ‘click-chemistry’
CN107226839B (zh) 一种rgd多肽偶联的酞菁硅光敏剂的合成和应用
Tewari et al. Peptide-targeted dendrimeric prodrugs of 5-aminolevulinic acid: A novel approach towards enhanced accumulation of protoporphyrin IX for photodynamic therapy
WO2020244570A1 (zh) 不同连接链偶联的叶酸靶向卟吩类光敏剂及其合成和应用
Hu et al. An advanced multifunctional prodrug combining photodynamic therapy with chemotherapy for highly efficient and precise tumor ablation
CN104718190A (zh) 用于光动力疗法的吡啶酮化合物
CN109265465B (zh) 一类新型焦脱镁叶绿酸a衍生物及其制备方法与应用
Fang et al. Smart glypican-3-targeting peptide–chlorin e6 conjugates for targeted photodynamic therapy of hepatocellular carcinoma
CN113717183B (zh) 周环不对称精氨酸修饰酞菁及其制备和在制药领域的应用
JPS625986A (ja) 新規なテトラピロ−ル化合物
EP3341022A2 (en) Conjugates
CN117700438A (zh) 一种具有高效光敏化产生羟基自由基的酞菁硅及其制备和应用
AU2002313562C1 (en) Compound
CN116173204A (zh) 一种谷胱甘肽激活的共组装纳米探针及其制备方法和应用
CN116135228A (zh) 一种竹红菌素2-位氨基取代或乙二胺取代的衍生物在制备抗肿瘤光动力药物中的应用
CN117384374A (zh) FTEP-TBFc化合物的制备方法和在抗肿瘤中的应用
JP4678661B2 (ja) 新規なテトラキスアミノフェニル（ジ）フェニレンジアミン骨格を有する化合物
AU2002313562A1 (en) Compound

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20818631

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 17/05/2022)

122 Ep: pct application non-entry in european phase

Ref document number: 20818631

Country of ref document: EP

Kind code of ref document: A1